WO2017049251A2 - Anticorps cd47 thérapeutiques - Google Patents

Anticorps cd47 thérapeutiques Download PDF

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Publication number
WO2017049251A2
WO2017049251A2 PCT/US2016/052383 US2016052383W WO2017049251A2 WO 2017049251 A2 WO2017049251 A2 WO 2017049251A2 US 2016052383 W US2016052383 W US 2016052383W WO 2017049251 A2 WO2017049251 A2 WO 2017049251A2
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Prior art keywords
seq
amino acid
acid sequence
light chain
heavy chain
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PCT/US2016/052383
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English (en)
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WO2017049251A3 (fr
Inventor
Pamela Manning
Robyn PURO
Juan C. Almagro
Robert W. KARR
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Tioma Therapeutics, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Priority to BR112018005322-8A priority Critical patent/BR112018005322A2/pt
Application filed by Tioma Therapeutics, Inc. filed Critical Tioma Therapeutics, Inc.
Priority to EP16847511.9A priority patent/EP3349787A4/fr
Priority to NZ740686A priority patent/NZ740686A/en
Priority to AU2016324316A priority patent/AU2016324316B2/en
Priority to UAA201808714A priority patent/UA126146C2/uk
Priority to CN202211086845.8A priority patent/CN116425875A/zh
Priority to RU2018124859A priority patent/RU2748401C2/ru
Priority to JP2018514882A priority patent/JP6885606B2/ja
Priority to CA2998644A priority patent/CA2998644A1/fr
Priority to MX2018003374A priority patent/MX2018003374A/es
Priority to CN201680062144.7A priority patent/CN108348589B/zh
Publication of WO2017049251A2 publication Critical patent/WO2017049251A2/fr
Publication of WO2017049251A3 publication Critical patent/WO2017049251A3/fr
Priority to US15/820,054 priority patent/US10239945B2/en
Priority to PH12018500515A priority patent/PH12018500515A1/en
Priority to IL258168A priority patent/IL258168A/en
Priority to ZA2018/04326A priority patent/ZA201804326B/en
Priority to US16/271,513 priority patent/US10844124B2/en
Priority to US17/084,156 priority patent/US20210070865A1/en

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Definitions

  • This disclosure is related generally to anti-CD47 monoclonal antibodies (anti-CD47 mAbs) with distinct functional profiles as described herein, methods to generate anti-CD47 mAbs, and to methods of using these anti-CD47 mAbs as therapeutics for the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury, cardiovascular diseases, autoimmune diseases, or inflammatory diseases or as diagnostics for determining the level of CD47 in tissue samples.
  • CD47 is a cell surface receptor comprised of an extracellular IgV set domain, a 5 transmembrane domain, and a cytoplasmic tail that is alternatively spliced.
  • Two ligands bind CD47: signal inhibitory receptor protein a (SIRPa) and thrombospondin-1 (TSP1).
  • SIRPa signal inhibitory receptor protein a
  • TSP1 thrombospondin-1
  • CD47 expression and/or activity have been implicated in a number of diseases and disorders. Accordingly, there exists a need for therapeutic compositions and methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, ischemia-reperfusion injury (IRI), cardiovascular diseases, or an autoimmune or inflammatory disease. There also exists a need for diagnostic compositions and methods for determining the level of CD47 expression in tumor samples.
  • anti-CD47 mAbs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPa; 3) increase phagocytosis of human tumor cells, 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) have reduced binding to human red blood cells (hRBCs); 7) have no detectable binding to hRBCs; 8) cause reduced agglutination of hRBCs; 9) cause no detectable agglutination of hRBCs; 10) reverse TSP1 inhibition of the nitric oxide (NO) pathway and/or 11) do not reverse TSP1 inhibition of the NO pathway.
  • NO nitric oxide
  • the antibodies of the disclosure are useful in various therapeutic methods for treating diseases and conditions associated with CD47 in humans and animals, including the prevention and treatment of solid and hematological cancers, autoimmune diseases, inflammatory diseases, IRI, and cardiovascular diseases.
  • the antibodies of the disclosure are also useful as diagnostics to determine the level of CD47 expression in tissue samples.
  • Embodiments of the disclosure include isolated antibodies and immunologically active binding fragments thereof; pharmaceutical compositions comprising one or more of the anti-CD47 monoclonal antibodies, preferably chimeric or humanized forms of said antibodies; methods of therapeutic use of such anti-CD47 monoclonal antibodies; and cell lines that produce these anti-CD47 monoclonal antibodies.
  • the embodiments of the disclosure include the mAbs, or antigen-binding fragments thereof, which are defined by reference to specific structural characteristics i.e. specified amino acid sequences of either the CDRs or entire heavy chain or light chain variable domains. All of these antibodies bind to CD47.
  • the monoclonal antibodies, or antigen binding fragments thereof may comprise at least one, usually at least three, CDR sequences as provided herein, usually in combination with framework sequences from a human variable region or as an isolated CDR peptide.
  • an antibody comprises at least one light chain comprising the three light chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a murine or human variable region framework, and at least one heavy chain comprising the three heavy chain CDR sequences provided herein situated in a variable region framework, which may be, without limitation, a human or murine variable region framework.
  • Preferred embodiments are anti-CD47 mAbs, or antigen binding fragments thereof, comprising a heavy chain variable domain comprising a variable heavy chain CDR1, variable heavy chain CDR2, and a variable heavy chain CDR3, wherein said variable heavy chain CDR1 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID N0:3; said variable heavy chain CDR2 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6; and said variable heavy chain CDR3 comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.
  • the heavy chain variable domain may comprise any one of the listed variable heavy chain
  • HCDR1 CDRl sequences in combination with any one of the variable heavy chain CDR2 sequences (HCDR2) and any one of the variable heavy chain CDR3 sequences (HCDR3).
  • HCDR2 and HCDR3 are particularly preferred, which derive from a single common VH domain, examples of which are described herein.
  • the antibody or antigen binding fragment thereof may additionally comprise a light chain variable domain (VL), which is paired with the VH domain to form an antigen binding domain.
  • VL light chain variable domain
  • Preferred light chain variable domains are those comprising a variable light chain CDRl, variable light chain CDR2, and a variable light chain CDR3, wherein said variable light chain CDRl comprises an amino acid sequence selected from the group consisting of:
  • variable light chain SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14; said variable light chain
  • CDR2 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17; and said variable light chain CDR3 optionally comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20.
  • the light chain variable domain may comprise any one of the listed variable light chain
  • LCDR1 in combination with any one of the variable light chain CDR2 sequences (LCDR2) and any one of the variable light chain CDR3 sequences (LCDR3).
  • LCDR2 variable light chain CDR2 sequences
  • LCDR3 variable light chain CDR3 sequences
  • certain embodiments of LCDR1 and LCDR2 and LCDR3 are particularly preferred, which derive from a single common VL domain, examples of which are described herein.
  • any given CD47 antibody or antigen binding fragment thereof comprising a VH domain paired with a VL domain will comprise a combination of 6 CDRs: variable heavy chain CDRl (HCDR1), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDRl (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDRl (LCDR1).
  • HCDR1 variable heavy chain CDRl
  • HCDR2 variable heavy chain CDR2
  • HCDR3 variable heavy chain CDR3
  • LCDR1 variable light chain CDR1
  • LCDR2 variable light chain CDR2
  • LCDR1 variable light chain CDR1
  • LCDR2 variable light chain CDR2
  • LCDR1 variable light chain CDR1
  • LCDR2 variable light chain CDR2
  • LCDR1 variable light chain CDR1
  • LCDR2 variable light chain CDR2
  • LCDR1 variable light chain CDR1
  • LCDR2 variable
  • Preferred combinations of 6 CDRs include, but are not limited to, the combinations of variable heavy chain CDRl (HCDRl), variable heavy chain CDR2 (HCDR2), variable heavy chain CDR3 (HCDR3), variable light chain CDRl (LCDR1), variable light chain CDR2 (LCDR2), and variable light chain CDR3 (LCDR3) selected from the group consisting of:
  • HCDRl comprising SEQ ID NO: l
  • HCDR2 comprising SEQ ID NO:4
  • HCDR3 comprising SEQ ID NO: 7, LCDR1 comprising SEQ ID NO: 11, LCDR2 comprising SEQ ID NO: 15, LCDR3 comprising SEQ ID NO: 18;
  • HCDRl comprising SEQ ID NO: l
  • HCDR2 comprising SEQ ID NO:4
  • LCDR3 compri sing SEQ ID NO : 18 ;
  • HCDRl comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO: 9, LCDR1 comprising SEQ ID NO: 12, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19;
  • HCDRl comprising SEQ ID NO:2, HCDR2 comprising SEQ ID NO:5, HCDR3 comprising SEQ ID NO: 9, LCDR1 comprising SEQ ID NO: 13, LCDR2 comprising SEQ ID NO: 16, LCDR3 comprising SEQ ID NO: 19; and
  • HCDRl comprising SEQ ID NO:3, HCDR2 comprising SEQ ID NO:6, HCDR3 comprising SEQ ID NO: 10, LCDR1 comprising SEQ ID NO: 14, LCDR2 comprising SEQ ID NO: 17, LCDR3 comprising SEQ ID NO: 20.
  • anti-CD47 antibodies include antibodies or antigen binding fragments thereof, comprising a heavy chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO: 32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, and SEQ ID NO:40 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.
  • preferred anti-CD47 antibodies including antibodies or antigen binding fragments thereof may comprise a light chain variable domain having an amino acid sequence selected from the group consisting of: the amino acid sequences of SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID N0:51, and SEQ ID NO:52 and amino acid sequences exhibiting at least 90%, 95%, 97%, 98%, or 99% sequence identity to one of the recited sequences.
  • preferred CD47 antibodies, or antigen binding fragments thereof are those comprising a combination of a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the combination is selected from the group consisting of:
  • Preferred anti-CD47 antibodies or antigen binding fragments thereof may also comprise a combination of a heavy chain variable domain and a light chain variable domain wherein the heavy chain variable domain comprises a VH sequence with at least 85% sequence identity, or at least 90%) sequence identity, or at least 95% sequence identity, or at least 97%, 98%> or 99% sequence identity, to the heavy chain amino acid sequences shown above in (i) to (xxxiv) and/or the light chain variable domain comprises a VL sequence with at least 85%> sequence identity, or at least 90%) sequence identity, or at least 95% sequence identity, or at least 97%, 98% or 99% sequence identity, to the light chain amino acid sequences shown above in (i) to (xxxiv).
  • the specific VH and VL pairings or combinations in parts (i) through (xxxiv) may be preserved for anti-CD47 antibodies having VH and VL domain sequences with a pailicular percentage sequence identity to these reference sequences.
  • the VH and/or VL domains may retain identical CDR sequences to those present in the reference sequence such that the variation is present only within the framework regions.
  • the preferred CD47 antibodies, or antigen binding fragments thereof are those comprising a combination of a heavy chain (HC) and a light chain (LC), wherein the combination is selected from the group consisting of:
  • VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
  • the preferred anti-CD47 antibodies described herein are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, the preferred anti-CD47 antibodies described herein are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the monoclonal antibody, or antigen binding fragment thereof specifically also binds to non-human primate CD47, wherein non-human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
  • the monoclonal antibody, or antigen binding fragment thereof binds to human, non-human primate, mouse, rabbit, and rat CD47.
  • the anti-CD47 mAbs disclosed can be full length humanized antibodies with human frameworks and constant regions of the isotypes, IgA, IgD, IgE, IgG, and IgM, more particularly, IgGl, IgG2, IgG3, IgG4, and in some cases with various mutations to alter Fc receptor function or prevent Fab arm exchangeor an antibody fragment, e.g., a F(ab') 2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc., as disclosed herein.
  • an antibody fragment e.g., a F(ab') 2 fragment, a F(ab) fragment, a single chain Fv fragment (scFv), etc.
  • compositions comprising one or more of the anti-CD47 mAbs or fragments disclosed herein, optionally chimeric or humanized forms, and a pharmaceutically acceptable carrier, diluent, or excipient.
  • anti-CD47 mAbs that possess the functional profiles as described herein.
  • the anti-CD47 mAbs of the present disclosure exhibit distinct combinations of properties, particularly combinations of properties that render the mAbs particularly advantageous or suitable for use in human therapy, particularly in the prevention and/or treatment of solid and hematological cancers, ischemia-reperfusion injury, autoimmune and/or inflammatory diseases.
  • FIG. 1A Binding of VLX4 Humanized mAbs to Human OV10 Cells Expressing human
  • VLX4 humanized mAbs VLX4hum_01 IgGl, VLX4hum_02 IgGl, VLX4hum_01 IgG4 PE, and VLX4hum_02 IgG4 PE
  • OV10 hCD47 OV10 cell line expressing human CD47
  • Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. IB Binding of VLX4 Humanized mAbs to Human OV10 Cells Expressing human CD47. Binding of VLX4 humanized mAbs (VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, and VLX4hum_13 IgG4 PE) to human CD47 was determined using an OV10 CD47 cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX4 representative mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • VLX4hum_06 IgG4 PE VLX4hum_07 IgG4 PE
  • VLX4hum_12 IgG4 PE VLX4hum_13 I
  • FIG. 2A Binding of VLX4 Humanized mAbs to Human RBCs (hRBCs). Binding of VLX4 humanized mAbs (VLX4hum_01 IgGl , VLX4hum_02 IgGl , VLX4hum_01 IgG4 PE, and VLX4hum_02 IgG4PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labeled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 2B Binding of VLX4 Humanized mAbs to Human RBCs. Binding of VLX4 humanized mAbs (VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, and VLX4hum_13 IgG4 PE) to human CD47 was determined using freshly isolated hRBCs. hRBCs were incubated for 60 minutes at 37°C with various concentrations of VLX4 mAbs, washed and incubated for lhr with FITC-labeled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • VLX4hum_07 IgG4 PE VLX4hum_12 IgG4 PE
  • VLX4hum_13 IgG4 PE VLX4hum_13 IgG4 PE
  • FIG. 3A Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells. Binding of
  • VLX8 IgG4PE chimera (xi) or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_04 IgG4 PE, VLX8hum_07 IgG4 PE, and VLX8hum_09 IgG4 PE) to human CD47 was determined using an OV10 hCD47 cell-based ELISA.
  • OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay.
  • Various concentrations of VLX8 representative mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 3B Binding of VLX8 Humanized mAbs to Human OV10 hCD47 Cells. Binding of VLX8 chimera or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2, and VLX8hum_09 IgG2) to human CD47 was determined using an OV10 hCD47 cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of VLX8 representative mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP-labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 4A Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8 IgG4PE xi or humanized mAbs (VLX8hum_01 IgG4PE, VLX8hum_03 IgG4PE, VLX8hum_07 IgG4PE, and VLX8hum_10 IgG4PE) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C with various concentrations of VLX8 mAbs, washed and incubated for lhr with FITC-labeled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 4B Binding of VLX8 Humanized mAbs to Human RBCs. Binding of VLX8
  • IgG4PE xi or humanized mAbs (VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 IgG2 and VLX8hum_09 IgG2) to human CD47 was determined using freshly isolated human RBCs. RBCs were incubated for 1 hr at 37°C with various concentrations of VLX8 mAbs, washed and incubated for lhr with FITC-labeled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 5A Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells.
  • VLX9 IgG2 xi or humanized mAbs (VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2 and VLX9hum_05 IgG2) to human CD47 was determined using an OV10 human CD47 cell-based ELISA.
  • OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay.
  • Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP -labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 5B Binding of VLX9 Humanized mAbs to Human OV10 hCD47 Cells. Binding of VLX9 IgG2 xi or humanized mAbs (VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, VLX9hum_09 IgG2 and VLX9hum_10 IgG2) to human CD47 was determined using a OV10 hCD47 cell-based ELISA. OV10 hCD47 cells were plated into 96 well plates and were confluent at the time of assay. Various concentrations of mAbs were added to the cells for 1 hr. Cells were washed and then incubated with HRP -labelled secondary antibody for 1 hr followed by addition of peroxidase substrate.
  • FIG. 6 Binding of VLX9 Humanized mAbs to Human RBCs. Binding of VLX9 IgG2 xi or humanized mAbs to human CD47 was determined using freshly isolated human hRBCs. RBCs were incubated for 60 minutes at 37°C with various concentrations of VLX9 mAbs, washed and incubated for lhr with FITC-labeled donkey anti-human antibody. Cells were washed and antibody binding measured using flow cytometry.
  • FIG. 7 VLX4, VLX8, and VLX9 Humanized mAbs Block SIRPa binding to CD47 on
  • Jurkat Cells 1.5 x 10 6 Jurkat cells were incubated with 5 ⁇ g/ml of VLX4, VLX8 and VLX9 CD47 humanized mAbs (VLX4hum_01 IgG4 PE, VLX4hum_07 IgG4 PE, VLX8hum_10 IgG4 PE, VLX4hum_l 1 IgG4 PE, VLX9hum_03 IgG2, VLX9hum_06 IgG2, and VLX9hum_08 IgG2) or a control antibody in RPMI containing 10% media for 30 min at 37°C. An equal volume of fluorescently labeled SIRPa-Fc fusion protein was added and incubated for an additional 30 min at 37°C. Cells were washed and binding was assessed using flow cytometry.
  • FIG. 8 VLX4 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat T Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE ( ⁇ ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of the VLX4 chimeric mAbs were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 9A VLX4 Humanized mAbs Increase Phagocytosis of Jurkat T Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of antibody were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non- phagocytosed Jurkat T cells were removed and macrophage cultures were washed extensively.
  • Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 9B VLX4 Humanized mAbs Increase Phagocytosis of Jurkat T Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of antibody were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non- phagocytosed Jurkat T cells were removed and macrophage cultures were washed extensively.
  • Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 10A VLX8 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat T Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of the VLX8 chimeric mAbs were added to the macrophage cultures and incubated at 37°C for 2 hrs. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD 14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 10B VLX8 Humanized mAbs Increase Phagocytosis of Jurkat Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hrs.
  • 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of antibody were added to the macrophage cultures and incubated at 37°C for 2 hrs.
  • Non- phagocytosed Jurkat T cells were removed and macrophage cultures were washed extensively.
  • Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14 + /CFSE + cells in the total CD14 + population.
  • FIG. 11A VLX9 CD47 Chimeric mAbs Increase Phagocytosis of Jurkat T Cells by Human Macrophages.
  • Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hours.
  • 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of the VLX9 chimeric mAbs were added to the macrophage cultures and incubated at 37°C for two hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
  • FIG. 11B VLX9 Humanized mAbs Increase Phagocytosis of Jurkat T Cells by Human
  • Macrophages Human macrophages were plated at a concentration of lxlO 4 cells per well in a 96 well plate and allowed to adhere for 24 hours. 5xl0 4 CFSE ( ⁇ ) labeled human Jurkat T cells and 1 ⁇ g/ml of antibody were added to the macrophage cultures and incubated at 37°C for two hours. Non-phagocytosed Jurkat cells were removed and macrophage cultures were washed extensively. Macrophages were trypsinized and stained for CD14. Flow cytometry was used to determine the percentage of CD14+/CFSE+ cells in the total CD14+ population.
  • FIG. 12A Induction of Cell Death in Human Jurkat T Cells by Soluble VLX4 Humanized mAbs.
  • Jurkat T cells (lxlO 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs in 1 ml of RPMI media for 24 hours at 37°C. Cells were then stained for annexin V and the signal was detected by flow cytometry.
  • FIG. 12B Induction of Cell Death in Human Jurkat T Cells by Soluble VLX4 Humanized mAbs.
  • Jurkat T cells (lxlO 4 ) were incubated with 1 ⁇ g/ml VLX4 humanized mAbs in 1 ml of RPMI media for 24 hours at 37°C. Cells were then stained for annexin V and the signal was detected by flow cytometry.
  • FIG. 13A Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 CD47
  • FIG. 13B Induction of Cell Death in Human Jurkat Cells by Soluble VLX8 Humanized mAbs.
  • Jurkat T ALL cells (1x104) were incubated with 1 ⁇ g/ml VLX8 humanized mAbs in 1 ml of RPMI media for 24 hrs at 37°C. Cells were then stained for annexin V and the signal was detected by flow cytometry.
  • FIG. 14A Induction of Cell Death of Human Jurkat Cells by Soluble VLX9 Murine/Human Chimeric mAbs.
  • lxlO 4 Jurkat cells were incubated with ⁇ g/ml of the VLX9 CD47 chimeric mAbs in 0.1 ml of RPMI media for 24 hours 37°C. Cells were then stained with annexin V and the signal analyzed by flow cytometry.
  • FIG. 14B Induction of Cell Death in Human Jurkat Cells by Soluble VLX9 Humanized mAbs.
  • Jurkat T ALL cells (lxlO 4 ) were incubated with 1 ⁇ g/ml VLX9 humanized mAbs in 1 ml of RPMI media for 24 hours at 37°C. Cells were then stained for annexin V and the signal was detected by flow cytometry.
  • VLX9 IgG2 (xi) is a murine/human chimera.
  • FIG. 15A Agglutination of hRBCs by VLX4 Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX4 mAbs (25 ⁇ g/mL - 0.4ng/mL). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 15B Agglutination of hRBCs by VLX8 Chimeric and Humanized mAbs. Hemagglutination was assessed following incubation of hRBCs with various concentrations of humanized VLX4 mAbs (25 ⁇ g/mL - 0.4ng/mL). Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. hRBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 ⁇ ) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 16 Agglutination of Human RBCs by VLX9 Humanized mAbs. Hemagglutination was assessed following incubation of human RBCs with various concentrations of VLX9 IgG2 chimera (xi) and humanized VLX9 mAbs. Blood was diluted (1 :50) and washed 3 times with PBS/EDTA/BSA. RBCs were added to U-bottomed 96 well plates with equal volumes of the antibodies (75 ⁇ 1) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • FIG. 17 VLX4 Humanized mAb Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70%) MatrigelTM (BD Biosciences; Bedford, MA) mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VLX4hum_07 or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 18 VLX8 Humanized mAb Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30% RPMI / 70%) MatrigelTM (BD Biosciences; Bedford, MA) mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measureed and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VLX8hum_10 or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 19 VLX9 Humanized mAb Reduces Tumor Growth in Raji Xenograft Model.
  • Female NSG mice were inoculated subcutaneously in the right flank with 0.1 mL of a 30%> RPMI / 70%) MatrigelTM (BD Biosciences; Bedford, MA) mixture containing a suspension of 5xl0 6 Raji tumor cells.
  • Five days following inoculation tumor volumes were measured and mice with palpable tumor volumes of 31-74 mm 3 were randomized into 8-10/group.
  • VLX9hum_08 IgG2 or PBS (control) administration was initiated at this time.
  • Mice were treated with 5 mg/kg of antibody 5X/week for 4 weeks by intraperitoneal injection. Tumor volumes and body weights were recorded twice weekly.
  • FIG. 20A Hemoglobin Levels in Blood following Administration of a Humanized VLX9 mAb to Cynomolgus Monkeys by Intravenous Infusion.
  • VLX9hum_08 IgG2 or vehicle were administered as a one hour intravenous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18. Hemoglobin levels were monitored throughout the study and normalized to control values.
  • FIG. 20B RBC Levels in Blood following Administration of Humanized VLX9 mAbs to Cynomolgus Monkeys by Intravenous Infusion.
  • VLX9hum_08 IgG2 or vehicle was administered as a one hour intraveneous infusion a dose of 5mg/kg on day 1 and a dose of 15mg/kg on day 18.
  • RBC levels were monitored throughout the study and normalized to control values.
  • FIG. 21 Immunohistochemical Staining of CD47 in Human Tumor Tissue with Anti- Murine/Rabbit Chimeric mAb.
  • CD47 was localized in human breast cancer tissue using VLX4 mouse/rabbit chimeric mAb. Paraffin-embedded tissue was sectioned, stained with 4 ug/ml of purified antibody and localized with anti-rabbit HRP secondary antibody. Arrows denote positive areas of CD47 staining.
  • FIG. 22 Summary of Anti-CD47 Antibody Properties.
  • CD47 integrated protein (IAP)
  • ovarian cancer antigen OA3 ovarian cancer antigen OA3
  • Rh-related antigen ovarian cancer antigen
  • anti-CD47 antibody refers to an antibody of the disclosure which is intended for use as a therapeutic or diagnostic agent, and therefore will typically possess the binding affinity required to be useful as a therapeutic and/or diagnostic agent.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • immunoglobulin immunoglobulin
  • immunoglobulin immunoglobulin
  • immunoglobulin molecules that contain an antigen binding site that specifically binds (immunoreacts with) an antigen.
  • specifically bind or “immunoreacts” with or directed against is meant that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds at a much lower affinity (Kd > 10 "6 ).
  • Antibodies include but are not limited to, polyclonal, monoclonal, chimeric, Fab fragments, Fab' fragments, F(ab') 2 fragments, single chain Fv fragments, and one-armed antibodies.
  • mAb monoclonal antibody
  • mAbs of the present disclosure preferably exist in a homogeneous or substantially homogeneous population. Complete mAbs contain 2 heavy chains and 2 light chains.
  • antibody fragment refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds.
  • antibody fragments include but are not limited to Fv, Fab, Fab', Fab' -SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formedfrom antibody fragments.
  • antibody compounds refers to mAbs and antigen-binding fragments thereof. Additional antibody compounds exhibiting similar functional properties according to the present disclosure can be generated by conventional methods. For example, mice can be immunized with human CD47 or fragments thereof, the resulting antibodies can be recovered and purified, and determination of whether they possess binding and functional properties similar to or the same as the antibody compounds disclosed herein can be assessed by the methods disclosed in Examples 3-11, below. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, chapters 5-8 and 15.
  • the monoclonal antibodies encompass antibodies in which a portion of the heavy and/or light chain is identical with, or homologous to, corresponding sequences in murine antibodies, in particular the murine CDRs, while the remainder of the chain(s) is (are) identical with, or homologous to, corresponding sequences in human antibodies.
  • Other embodiments of the disclosure include antigen-binding fragments of these monoclonal antibodies that exhibit binding and biological properties similar or identical to the monoclonal antibodies.
  • the antibodies of the present disclosure can comprise kappa or lambda light chain constant regions, and heavy chain IgA, IgD, IgE, IgG, or IgM constant regions, including those of IgG subclasses IgGl, IgG2, IgG3, and IgG4 and in some cases with various mutations to alter Fc receptor function.
  • the monoclonal antibodies containing the presently disclosed murine CDRs can be prepared by any of the various methods known to those skilled in the art, including recombinant DNA methods.
  • a full-length antibody as it exists naturally is a "Y" shaped immunoglobulin (Ig) molecule comprising four polypeptide chains: two identical heavy (H) chains and two identical light (L) chains, interconnected by disulfide bonds.
  • the amino terminal portion of each chain termed the fragment antigen binding region (FAB), includes a variable region of about 100-110 or more amino acids primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein.
  • CDRs complementarity determining regions
  • the carboxy -terminal portion of each chain defines a constant region (the "Fc" region) primarily responsible for effector function.
  • Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the 3 CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3 " and the 3 CDRs of the heavy chain are referred to as "HCDRl, HCDR2, and HCDR3.
  • the CDRs contain most of the residues which form specific interactions with the antigen.
  • the "antigen-binding site” can also be defined as the "Hypervariable regions", “HVRs”, or “HVs”, and refer to the structurally hypervariable regions of antibody variable domains as defined by Chothia and Lesk (Chothia and Lesk, Mol. Biol. 196:901-917, 1987).
  • HVRs HVRs
  • VL VL
  • CDRs as defined by Kabat except in H-CDR1, which is extended to include HI .
  • Ig heavy chains There are five types of mammalian immunoglobulin (Ig) heavy chains, denoted by the Greek letters a (alpha), ⁇ (delta), ⁇ (epsilon), ⁇ (gamma), and ⁇ (mu), which define the class or isotype of an antibody as IgA, IgD, IgE, IgG, or IgM, respectively.
  • IgG antibodies can be further divided into subclasses, for example, IgGl, IgG2, IgG3, and IgG4.
  • Each heavy chain type is characterized by a particular constant region with a sequence well known in the art.
  • the constant region is identical in all antibodies of the same isotype, but differs in antibodies of different isotypes.
  • Heavy chains ⁇ , a, and ⁇ have a constant region composed of three tandem immunoglobulin (Ig) domains, and a hinge region for added flexibility.
  • Heavy chains ⁇ and ⁇ have a constant region composed of four Ig domains.
  • the hinge region is a flexible amino acid stretch that links the Fc and Fab portions of an antibody. This regions contains cysteine residues that can form disulfide bonds, connecting two heavy chains together.
  • variable region of the heavy chain differs in antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone.
  • the variable region of each heavy chain is approximately 1 10 amino acids long and is composed of a single Ig domain.
  • light chains are classified as kappa ( ⁇ ) or lambda ( ⁇ ), and are characterized by a particular constant region as known in the art.
  • a light chain has two successive domains: one variable domain at the amino-terminal end, and one constant domain at the carboxy -terminal end.
  • Each antibody contains two light chains that are always identical; only one type of light chain, ⁇ or ⁇ , is present per antibody in mammals.
  • the Fc region composed of two heavy chains that contribute three or four constant domains depending on the class of the antibody, plays a role in modulating immune cell activity. By binding to specific proteins, the Fc region ensures that each antibody generates an appropriate immune response for a given antigen.
  • the Fc region also binds to various cell receptors, such as Fc receptors, and other immune molecules, such as complement proteins. By doing this, it mediates different physiological effects, including opsonization, cell lysis, and degranulation of mast cells, basophils and eosinophils.
  • epitope refers to a specific arrangement of amino acids located on a peptide or protein to which an antibody or antibody fragment binds.
  • Epitopes often consist of a chemically active surface grouping of molecules such as amino acids or sugar side chains, and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • Epitopes can be linear, i.e., involving binding to a single sequence of amino acids, or conformational, i.e., involving binding to two or more sequences of amino acids in various regions of the antigen that may not necessarily be contiguous in the linear sequence.
  • the terms “specifically binds”, “bind specifically”, “specific binding”, and the like as applied to the present antibody compounds refer to the ability of a specific binding agent (such as an antibody) to bind to a target molecular species in preference to binding to other molecular species with which the specific binding agent and target molecular species are admixed.
  • a specific binding agent is said specifically to recognize a target molecular species when it can bind specifically to that target.
  • binding affinity refers to the strength of binding of one molecule to another at a site on the molecule. If a particular molecule will bind to or specifically associate with another particular molecule, these two molecules are said to exhibit binding affinity for each other. Binding affinity is related to the association constant and dissociation constant for a pair of molecules, but it is not critical to the methods herein that these constants be measured or determined.
  • affinities as used herein to describe interactions between molecules of the described methods are generally apparent affinities (unless otherwise specified) observed in empirical studies, which can be used to compare the relative strength with which one molecule (e.g., an antibody or other specific binding partner) will bind two other molecules (e.g., two versions or variants of a peptide).
  • one molecule e.g., an antibody or other specific binding partner
  • two other molecules e.g., two versions or variants of a peptide.
  • sequence identity means the percentage of identical nucleotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in: Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, by the homology alignment algorithms, by the search for similarity method or, by computerized implementations of these algorithms (GAP, BESTFIT, PASTA, and TFASTA in the GCG Wisconsin Package, available from Accelrys, Inc., San Diego, California, United States of America), or by visual inspection. See generally, Altschul, S. F. et al., J. Mol. Biol. 215: 403-410 (1990) and Altschul et al. Nucl. Acids Res. 25: 3389-3402 (1997).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in (Altschul, S., et al., NCBI LM NIH Bethesda, Md. 20894; and Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always; 0) and N (penalty score for mismatching residues; always; 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value, the cumulative score goes to zero or below due to the accumulation of one or more negative-scoring residue alignments, or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences.
  • test nucleic acid sequence is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid sequence to the reference nucleic acid sequence is in one embodiment less than about 0.1, in another embodiment less than about 0.01, and in still another embodiment less than about 0.001.
  • the terms “humanized”, “humanization”, and the like refer to grafting of the murine monoclonal antibody CDRs disclosed herein to human FRs and constant regions. Also encompassed by these terms are possible further modifications to the murine CDRs, and human FRs, by the methods disclosed in, for example, Kashmiri et al. (2005) Methods 36(l):25-34 and Hou et al. (2008) J. Biochem. 144(1): 115-120, respectively, to improve various antibody properties, as discussed below.
  • humanized antibodies refers to mAbs and antigen binding fragments thereof, including the antibody compounds disclosed herein, that have binding and functional properties according to the disclosure similar to those disclosed herein, and that have FRs and constant regions that are substantially human or fully human surrounding CDRs derived from a non-human antibody.
  • FR or "framework sequence” refers to any one of FRs 1 to 4.
  • Humanized antibodies and antigen binding fragments encompassed by the present disclosure include molecules wherein any one or more of FRs 1 to 4 is substantially or fully human, i.e., wherein any of the possible combinations of individual substantially or fully human FRs 1 to 4, is present.
  • this includes molecules in which FR1 and FR2, FR1 and FR3, FR1, FR2, and FR3, etc., are substantially or fully human.
  • Substantially human frameworks are those that have at least 80% sequence identity to a known human germline framework sequence.
  • the substantially human frameworks have at least 85%>, at least 86%>, at least 87%>, at least 88%>, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%), at least 97%>, at least 98%>, or at least 99%> sequence identity, to a framework sequence disclosed herein, or to a known human germline framework sequence.
  • Fully human frameworks are those that are identical to a known human germline framework sequence.
  • Human FR germline sequences can be obtained from the international ImMunoGeneTics (IMGT) database and from The Immunoglobulin FactsBook by Marie-Paule Lefranc and Gerard Lefranc, Academic Press, 2001, the contents of which are herein incorporated by reference in their entirety.
  • IMGT international ImMunoGeneTics
  • the Immunoglobulin Facts Book is a compendium of the human germline immunoglobulin genes that are used to create the human antibody repertoire, and includes entries for 203 genes and 459 alleles, with a total of 837 displayed sequences.
  • the individual entries comprise all the human immunoglobulin constant genes, and germline variable, diversity, and joining genes that have at least one functional or open reading frame allele, and which are localized in the three major loci.
  • germline light chain FRs can be selected from the group consisting of: IGKV3D-20, IGKV2-30, IGKV2-29, IGKV2-28, IGKV1-27, IGKV3-20, IGKV1-17, IGKV1-16, 1-6, IGKV1- 5, IGKV1-12, IGKV1D-16, IGKV2D-28, IGKV2D-29, IGKV3-11, IGKV1-9, IGKV1-39, IGKV1D-39 and IGKV1D-33 and IGKJ1-5 and germline heavy chain FRs can be selected from the group consisting of: IGHV1-2, IGHV1-18, IGHV1-46, IGHV1-69, IGHV2-5, IGHV2-26, IGHV2-70, IGHV1-3, IGHV1-8, IGHV3-9, IGHV3-11, IGHV3- 15, IGHV3-20
  • Substantially human FRs are those that have at least 80% sequence identity to a known human germline FR sequence.
  • the substantially human frameworks have at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, to a framework sequences disclosed herein, or to a known human germline framework sequence.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having sequence identities of at least 80%, at least 85%, at least 90%), at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to a CDR sequence disclosed herein.
  • CDRs encompassed by the present disclosure include not only those specifically disclosed herein, but also CDR sequences having 1, 2, 3, 4, or 5 amino acid changes at corresponding positions compared to CDR sequences disclosed herein.
  • Such sequence identical, or amino acid modified, CDRs preferably bind to the antigen recognized by the intact antibody.
  • Humanized antibodies in addition to those disclosed herein exhibiting similar functional properties according to the present disclosure can be generated using several different methods Almagro et al. Frontiers in Biosciences. Humanization of antibodies. (2008) Jan 1; 13 : 1619-33.
  • the parent antibody compound CDRs are grafted into a human framework that has a high sequence identity with the parent antibody compound framework.
  • sequence identity of the new framework will generally be at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identical to the sequence of the corresponding framework in the parent antibody compound.
  • frameworks having fewer than 100 amino acid residues one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody.
  • the framework can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in vicieri et al. Bioinformatics. 2015 Feb l;31(3):434-435 and U.S. Patents 4,816,397, 5,225,539, and 5,693,761; and the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239: 1534-1536.
  • the parent antibody compound CDRs are grafted into a human FR that has a high sequence identity with the parent antibody compound framework.
  • the sequence identity of the new FR will generally be at least 80%, at least 85%, at least 90%, at least 95%, at least 96%), at least 97%, at least 98%, or at least 99% identical to the sequence of the corresponding FR in the parent antibody compound.
  • FRs having fewer than 100 amino acid residues one, two, three, four, five, or more amino acid residues can be changed. This grafting may result in a reduction in binding affinity compared to that of the parent antibody.
  • the FR can be back-mutated to the parent framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references describing methods useful to generate humanized variants based on homology and back mutations include as described in vicieri et al. Bioinformatics. 2015 Feb l;31(3):434-435 and U.S. Patents 4,816,397, 5,225,539, and 5,693,761; and the method of Winter and co-workers (Jones et al. (1986) Nature 321 :522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. ( ⁇ 9 ) Science 239: 1534-1536.
  • any side chain atom of a framework amino acid is within about 5-6 angstroms (center- to-center) of any atom of a CDR amino acid in a three dimensional immunoglobulin model.
  • Another approach to generating humanized antibodies exhibiting similar functional properties to the antibody compounds disclosed herein involves randomly mutating amino acids within the grafted CDRs without changing the framework, and screening the resultant molecules for binding affinity and other functional properties that are as good as, or better than, those of the parent antibody compounds.
  • Single mutations can also be introduced at each amino acid position within each CDR, followed by assessing the effects of such mutations on binding affinity and other functional properties.
  • Single mutations producing improved properties can be combined to assess their effects in combination with one another. Further, a combination of both of the foregoing approaches is possible.
  • After CDR grafting one can back-mutate specific FRs in addition to introducing amino acid changes in the CDRs. This methodology is described in Wu et al. (1999) J. Mol Biol. 294: 151-162.
  • amino acid substitution within the frameworks is restricted to one, two, three, four, or five positions within any one or more of the four light chain and/or heavy chain FRs disclosed herein.
  • amino acid substitution within the CDRs is restricted to one, two, three, four, or five positions within any one or more of the three light chain and/or heavy chain CDRs. Combinations of the various changes within these FRs and CDRs described above are also possible.
  • HAMA murine antibodies have been genetically manipulated to progressively replace their murine content with the amino acid residues present in their human counterparts by grafting their complementarity determining regions (CDRs) onto the variable light (VL) and variable heavy (VH) frameworks of human immunoglobulin molecules, while retaining those murine framework residues deemed essential for the integrity of the antigen-combining site.
  • CDRs complementarity determining regions
  • VL variable light
  • VH variable heavy
  • the xenogeneic CDRs of the humanized antibodies may evoke anti-idiotypic (anti-Id) response in patients.
  • SDR grafting a procedure to humanize xenogeneic antibodies by grafting onto the human frameworks only the CDR residues most crucial in the antibody-ligand interaction, called "SDR grafting", has been developed, wherein only the crucial specificity determining residues (SDRs) of CDRS are grafted onto the human frameworks.
  • SDR grafting involves identification of SDRs through the help of a database of the three-dimensional structures of the antigen-antibody complexes of known structures, or by mutational analysis of the antibody-combining site.
  • Embodiments of the present disclosure encompass antibodies created to avoid recognition by the human immune system containing CDRs disclosed herein in any combinatorial form such that contemplated mAbs can contain the set of CDRs from a single murine mAb disclosed herein, or light and heavy chains containing sets of CDRs comprising individual CDRs derived from two or three of the disclosed murine mAbs.
  • Such mAbs can be created by standard techniques of molecular biology and screened for desired activities using assays described herein. In this way, the disclosure provides a "mix and match" approach to create novel mAbs comprising a mixture of CDRs from the disclosed murine mAbs to achieve new, or improved, therapeutic activities.
  • Monoclonal antibodies or antigen-binding fragments thereof encompassed by the present disclosure that "compete" with the molecules disclosed herein are those that bind human CD47 at site(s) that are identical to, or overlapping with, the site(s) at which the present molecules bind. Competing monoclonal antibodies or antigen-binding fragments thereof can be identified, for example, via an antibody competition assay. For example, a sample of purified or partially purified human CD47 extracellular domain can be bound to a solid support. Then, an antibody compound, or antigen binding fragment thereof, of the present disclosure and a monoclonal antibody or antigen-binding fragment thereof suspected of being able to compete with such disclosure antibody compound are added. One of the two molecules is labeled.
  • the labeled compound and the unlabeled compound bind to separate and discrete sites on CD47, the labeled compound will bind to the same level whether or not the suspected competing compound is present. However, if the sites of interaction are identical or overlapping, the unlabeled compound will compete, and the amount of labeled compound bound to the antigen will be lowered. If the unlabeled compound is present in excess, very little, if any, labeled compound will bind.
  • competing monoclonal antibodies or antigen-binding fragments thereof are those that decrease the binding of the present antibody compounds to CD47 by about 50%, about 60%, about 70%, about 80%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99%.
  • Details of procedures for carrying out such competition assays are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. Such assays can be made quantitative by using purified antibodies.
  • a standard curve is established by titrating one antibody against itself, i.e., the same antibody is used for both the label and the competitor.
  • the capacity of an unlabeled competing monoclonal antibody or antigen-binding fragment thereof to inhibit the binding of the labeled molecule to the plate is titrated. The results are plotted, and the concentrations necessary to achieve the desired degree of binding inhibition are compared.
  • mAbs or antigen-binding fragments thereof that compete with antibody compounds of the present disclosure in such competition assays possess the same or similar functional properties of the present antibody compounds can be determined via these methods in conjunction with the methods described in Examples 3-5, below.
  • competing antibodies possess about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%), about 99%), or identical biological activity compared to that of the antibody compounds disclosed herein as determined by the methods disclosed in the Examples presented below.
  • the mAbs or antigen-binding fragments thereof, or competing antibodies useful in the compositions and methods can be any of the isotypes described herein. Furthermore, any of these isotypes can comprise further amino acid modifications as follows.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgGl isotype.
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter antibody half-life.
  • Antibody half-life is regulated in large part by Fc-dependent interactions with the neonatal Fc receptor (Roopenian and Alikesh, 2007).
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody can be modified to increase half-life include, but are not limited to amino acid modifications N434A, T307A/E380A/N434A (Petkova et al., 2006, Yeung et al., 2009); M252Y/S254T/T256E (Dall'Acqua et al., 2006); T250Q/M428L (Hinton et al., 2006); and M428L/N434S (Zalevsky et al., 2010).
  • amino acid modifications N434A, T307A/E380A/N434A Petkova et al., 2006, Yeung et al., 2009
  • M252Y/S254T/T256E Dall'Acqua et al., 2006
  • T250Q/M428L Hinton et al., 2006
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease half-life and/or decrease endogenous IgG include, but are not limited to amino acid modifications I253A (Petkova et al., 2006); P257I/N434H, D376V/N434H (Datta-Mannan et al., 2007); and M252Y/S254T/T256E/H433K/N434F (Vaccaro et al., 2005).
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody- Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody -Dependent Cellular Phagocytosis (ADCP), Clq binding, and altered binding to Fc receptors.
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to amino acid modifications S298A/E333A/K334 (Shields et al., 2001); S239D/I332E and S239D/A330L/I332E (Lazar et al., 2006); F234L/R292P/Y300L, F234L/R292P/Y300L/P393L, and F243L/R292P/Y300L/V305I/P396L (Stevenhagen et al., 2007); G236A, G236A/S239D/I332E, and G236A/S239D/A330L/I332E (Richards et al., 2008); K326A/E333A, K326A/E333S and K326W/E333S
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications N297A and N297Q (Bolt et al., 1993, Walker et al., 1989); L234A/L235A (Xu et al., 2000); K214T/E233P/L234V/L235A/G236- deleted/A327G/P331A/D356E/L358M (Ghevaert et al., 2008);
  • the human IgGl constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331 S, H268Q/A330S/V309L/P331 S, H268D/A330S/V309L/P331 S, H268Q/A330R/V309L/P331 S, H268D/A330R/V309L/P331 S, H268D/A330R/V309L/P331 S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G
  • P238D/E233D/P271G/A330S P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S,
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG2 isotype.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase or decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody- Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), Antibody -Dependent Cellular Phagocytosis (ADCP), and Clq binding, and altered binding to Fc receptors.
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to increase antibody effector function include, but are not limited to the amino acid modification K326A/E333S (Idusogie et al., 2001).
  • the human IgG2 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector function include, but are not limited to amino acid modifications V234A/G237A (Cole et al., 1999); E233D, G237D, P238D, H268Q, H268D, P271G, V309L, A330S, A330R, P331 S, H268Q/A330S/V309L/P331 S, H268D/A330S/V309L/P331 S, H268Q/A330R/V309L/P331 S, H268D/A330R/V309L/P331 S, H268D/A330R/V309L/P331 S, E233D/A330R, E233D/A330S, E233D/P271G/A330R, E233D/P271G/A330S, G
  • P238D/E233D/P271G/A330S P238D/G237D/H268D/P271G, P238D/G237D/H268Q/P271G, P238D/G237D/ P271G/A330R, P238D/G237D/ P271G/A330S,
  • P238D/E233D/H268D/P271G/A330R P238D/E233D/H268Q/P271G/A330R
  • P238D/E233D/H268D/P271G/A330S P238D/E233D/H268Q/P271G/A330S
  • P238D/G237D/H268D/P271 G/A33 OR, P238D/G237D/H268Q/P271 G/A33 OR, P238D/G237D/H268D/P271 G/A330 S, P238D/G237D/H268Q/P271 G/A330 S,
  • the Fc region of a human IgG2 of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to alter isoform and/or agonistic activity, include, but are not limited to amino acid modifications C127S (CHI domain), C232S, C233S, C232S/C233S, C236S, and C239S (White et al., 2015, Lightle et al., 2010).
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG3 isotype.
  • the human IgG3 constant region of the monoclonal antibody, or antigen binding fragment thereof wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at one or more amino acid(s) to increase antibody half-life, Antibody-Dependent Cellular Cytotoxicity (ADCC), Complement-Dependent Cytotoxicity (CDC), or apoptosis activity.
  • ADCC Antibody-Dependent Cellular Cytotoxicity
  • CDC Complement-Dependent Cytotoxicity
  • apoptosis activity apoptosis activity.
  • the human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof, wherein said human IgG3 constant region of the monoclonal antibody, or antigen-binding fragment thereof can be modified at amino acid R435H to increase antibody half-life.
  • the monoclonal antibody or antigen-binding fragment thereof, or competing antibody described herein can be of the human IgG4 isotype.
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to decrease antibody effector functions.
  • antibody effector functions include, but are not limited to, Antibody -Dependent Cellular Cytotoxicity (ADCC) and Antibody-Dependent Cellular Phagocytosis (ADCP).
  • the human IgG4 constant region of the monoclonal antibody, antigen-binding fragment thereof, or competing antibody described herein can be modified to prevent Fab arm exchange and/or decrease antibody effector function include, but are not limited to amino acid modifications F234A/L235A (Alegre et al., 1994); S228P, L235E and S228P/L235E (Reddy et al., 2000).
  • tumor refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues.
  • cancer cancer
  • cancer cancer and cancerrous refer to or describe the physiological condition in mammals that is typically characterized by aberrant cell growth/proliferation.
  • cancers include, but are not limited to, carcinoma, lymphoma (i.e., Hodgkin's and non-Hodgkin's lymphoma), blastoma, sarcoma, and leukemia.
  • cancers include squamous cell cancer, small-cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastrointestinal cancer, pancreatic cancer, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney cancer, liver cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, leukemia and other lymphoproliferative disorders, and various types of head and neck cancer.
  • susceptible cancer refers to a cancer, cells of which express
  • CD47 are responsive to treatment with an antibody or antigen binding fragment thereof, or competing antibody or antigen binding fragment thereof, of the present disclosure.
  • autoimmune disease refers to when the body's immune system turns against itself and mistakenly attacks healthy cells.
  • inflammatory disease refers to a disease characterized by inflammation which is a fundamental pathologic process consisting of a dynamic complex of histologically apparent cytologic changes, cellular infiltration, and mediator release that occurs in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent, including the local reactions and resulting morphologic changes; the destruction or removal of the injurious material; and the responses that lead to repair and healing.
  • autoinflammatory disease refers to a disease that results when the innate immune system causes inflammation for unknown reasons.
  • ischemia refers to a vascular phenomenon in which a decrease in the blood supply to a bodily organ, tissue, or part is caused, for instance, by constriction or obstruction of one or more blood vessels. Ischemia sometimes results from vasoconstriction or thrombosis or embolism. Ischemia can lead to direct ischemic injury, tissue damage due to cell death caused by reduced oxygen supply. Ischemia can occur acutely, as during surgery, or from trauma to tissue incurred in accidents, injuries and war settings, or following harvest of organs intended for subsequent transplantation, for example. It can also occur sub-acutely, as found in atherosclerotic peripheral vascular disease, where progressive narrowing of blood vessels leads to inadequate blood flow to tissues and organs.
  • ischemia When a tissue is subjected to ischemia, a sequence of chemical events is initiated that may ultimately lead to cellular dysfunction and necrosis. If ischemia is ended by the restoration of blood flow, a second series of injurious events ensue, producing additional injury.
  • the resultant injury involves two components—the direct injury occurring during the ischemic interval, and the indirect or reperfusion injury that follows.
  • Ischemic stroke can be caused by several different kinds of diseases. The most common problem is narrowing of the arteries in the neck or head. This is most often caused by atherosclerosis, or gradual cholesterol deposition. If the arteries become too narrow, blood cells may collect in them and form blood clots (thrombi). These blood clots can block the artery where they are formed (thrombosis), or can dislodge and become trapped in arteries closer to the brain (embolism). Cerebral stroke can occur when atherosclerotic plaque separates away partially from the vessel wall and occludes the flow of blood through the blood vessel.
  • Reperfusion refers to restoration of blood flow to tissue that is ischemic, due to decrease in blood flow.
  • Reperfusion is a procedure for treating infarction or other ischemia, by enabling viable ischemic tissue to recover, thus limiting further necrosis.
  • reperfusion can itself further damage the ischemic tissue, causing reperfusion injury.
  • ischemic/reperfusion injury involves tissue injury that occurs after blood flow is restored. Current understanding is that much of this injury is caused by chemical products, free radicals, and active biological agents released by the ischemic tissues.
  • Nitric oxide (NO) donor, precursor, or nitric oxide generating topical agent refers to a compound or agent that either delivers NO, or that can be converted to NO through enzymatic or non-enzymatic processes. Examples include, but are not limited to, NO gas, isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetyl- penicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil ® , and arginine.
  • NO gas isosorbide dinitrite, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S-nitroso-N-acetyl- penicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothio
  • Soluble guanylyl cyclase is the receptor for nitric oxide in vascular smooth muscle.
  • nitric oxide is endogenously generated by endothelial nitric oxide synthase from L-arginine, and activates soluble guanylyl cyclase in adjacent vascular smooth muscle cells to increase cGMP levels, inducing vascular relaxation.
  • Nitric oxide binds to the normally reduced heme moiety of soluble guanylyl cyclase, and increases the formation of cGMP from GTP, leading to a decrease in intracellular calcium, vasodilation, and anti-inflammatory effects.
  • Oxidation of the heme iron on sGC decreases responsiveness of the enzyme to nitric oxide, and promotes vasoconstriction.
  • the nitric oxide-sGC-cGMP pathway therefore plays an important role in cardiovascular diseases.
  • Nitrogen-containing compounds such as sodium azide, sodium nitrite, hydroxylamine, nitroglycerin, and sodium nitroprusside have been shown to stimulate sGC, causing an increase in cGMP, and vascular relaxation.
  • activators of sGC activate the oxidized or heme-deficient sGC enzyme that is not responsive to nitric oxide, i.e., they stimulate sGC independent of redox state.
  • stimulators of of sGC can enhance the sensitivity of reduced sGC to nitric oxide
  • activators of sGC can increase sGC enzyme activity even when the enzyme is oxidized and is therefore less, or unresponsive, to nitric oxide.
  • sGC activators are non-nitric oxide based.
  • An agent that activates soluble guanylyl cyclase refers, for example, to organic nitrates (Artz et al. (2002) J. Biol. Chem. 277: 18253-18256); protoporphyrin IX (Ignarro et al. ⁇ 9%2) Proc. Natl. Acad. Sci. USA 79:2870-2873); YC-1 (Ko et al. (1994) Blood 84:4226-4233); BAY 41-2272 and BAY 41-8543 (Stasch et al. (2001 Nature 410 (6825): 212-5), CMF-1571, and A-350619 (reviewed in Evgenov et al. (2006) Nat.
  • cGMP can also be increased by inhibiting degradation using phosphodiesterase inhibitors.
  • an agent that inhibits cyclic nucleotide phosphodiesterases include, tadalafil, vardenafil, udenafil, and sildenafil avanafil.
  • term “treating” or “treat” or “treatment” means slowing, interrupting, arresting, controlling, stopping, reducing, or reversing the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.
  • the term “treating” and the like refer to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • an antibody compound of the present disclosure which, upon single or multiple dose administration to a patient or organ, provides the desired treatment or prevention.
  • Therapeutically effective amounts of the present antibody compounds can also comprise an amount in the range of from about 0.1 mg/kg to about 150 mg/kg, from about 0.1 mg/kg to about 100 mg/kg, from about 0.1 mg/kg to about 50 mg/kg, or from about 0.05 mg/kg to about 10 mg/kg per single dose administered to a harvested organ or to a patient.
  • Known antibody-based pharmaceuticals provide guidance in this respect.
  • HerceptinTM is administered by intravenous infusion of a 21 mg/ml solution, with an initial loading dose of 4 mg/kg body weight and a weekly maintenance dose of 2 mg/kg body weight; RituxanTM is administered weekly at 375 mg/m 2 ; for example.
  • a therapeutically effective amount for any individual patient can be determined by the health care provider by monitoring the effect of the antibody compounds on tumor regression, circulating tumor cells, tumor stem cells or anti-tumor responses. Analysis of the data obtained by these methods permits modification of the treatment regimen during therapy so that optimal amounts of antibody compounds of the present disclosure, whether employed alone or in combination with one another, or in combination with another therapeutic agent, or both, are administered, and so that the duration of treatment can be determined as well. In this way, the dosing/treatment regimen can be modified over the course of therapy so that the lowest amounts of antibody compounds used alone or in combination that exhibit satisfactory efficacy are administered, and so that administration of such compounds is continued only so long as is necessary to successfully treat the patient.
  • Known antibody-based pharmaceuticals provide guidance relating to frequency of administration e.g., whether a pharmaceutical should be delivered daily, weekly, monthly, etc. Frequency and dosage may also depend on the severity of symptoms.
  • antibody compounds of the present disclosure can be used as medicaments in human and veterinary medicine, administered by a variety of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intratumoral, intranasal, enteral, sublingual, intravaginal, intravesiciular or rectal routes.
  • the compositions can also be administered directly into a lesion such as a tumor. Dosage treatment may be a single dose schedule or a multiple dose schedule. Hypo sprays may also be used to administer the pharmaceutical compositions.
  • the therapeutic compositions can be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • Veterinary applications include the treatment of companion/pet animals, such as cats and dogs; working animals, such as guide or service dogs, and horses; sport animals, such as horses and dogs; zoo animals, such as primates, cats such as lions and tigers, bears, etc.; and other valuable animals kept in captivity.
  • compositions can be prepared by methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21 st Edition (2005), Lippincott Williams & Wilkins, Philadelphia, PA, and comprise one or more antibody compounds disclosed herein, and a pharmaceutically or veterinarily acceptable, for example, physiologically acceptable, carrier, diluent, or excipient.
  • anti-CD47 mAbs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPa; 3) increase phagocytosis of human tumor cells, 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) have reduced binding to human red blood cells (hRBCs); 7) have no detectable binding to hRBCs; 8) cause reduced agglutination of hRBCs; 9) cause no detectable agglutination of hRBCs; 10) reverse TSP1 inhibition of the nitric oxide (NO) pathway and/or 11) do not reverse TSP1 inhibition of the NO pathway.
  • NO nitric oxide
  • anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure possess combinations of properties that are distinct from the anti-CD47 antibodies of the prior art. These properties and characteristics will now be described in further detail.
  • the anti-CD47 antibodies, and antigen binding fragments thereof, of the present disclosure bind human CD47.
  • the anti-CD47 antibodies exhibit cross-reactivity with one or more species homologs of CD47, for example CD47 homologs of non-human primate origin.
  • the anti-CD47 antibodies and antigen binding fragments thereof of the present disclosure bind to human CD47 and to CD47 of non-human primate, mouse, rat, and/or rabbit origin.
  • the cross-reactivity with other species homologs can be particularly advantageous in the development and testing of therapeutic antibodies.
  • pre-clinical toxicology testing of therapeutic antibodies is frequently carried out in non-human primate species including, but not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
  • Cross-reactivity with these species homologs can therefore be particularly advantageous for the development of antibodies as clinical candidates.
  • CD47 also known as integrin associated protein (IAP) is a 50 kDa cell surface receptor that is comprised of an extracellular N-terminal IgV domain, a five membrane spanning transmembrane domain, and a short C-terminal intracellular tail that is alternatively spliced.
  • IAP integrin associated protein
  • SIRPa Signal Regulatory Protein alpha
  • TSP1 Thrombospondin-1
  • SIRPa on a phagocyte engages CD47 on a target cell
  • this interaction prevents phagocytosis of the target cell.
  • the interaction of CD47 and SIRPa effectively sends a "don't eat me” signal to the phagocyte (Oldenborg et al. Science 288: 2051-2054, 2000).
  • Blocking the interaction of SIRPa and CD47 with an anti-CD47 mAb in a therapeutic context can provide an effective anti-cancer treatment by promoting the uptake and clearance of cancer cells by the host' s immune system.
  • an important functional characteristic of some anti-CD47 mAbs is the ability to block the interaction of CD47 and SIRPa, resulting in phagocytosis of CD47 expressing tumor cells by macrophages.
  • B6H12 and BRIC126 have also been shown to cause phagocytosis of human tumor cells by human and mouse macrophages (Willingham et al. Proc Natl Acad Sci USA 109(17):6662-6667, 2012; Chao et al. Cell 142:699-713, 2012; EP 2 242 512 Bl).
  • the term "blocks SIRPa binding to human CD47” refers to a greater than 50% reduction of SIRPa-Fc binding to CD47 on Jurkat cells by an anti-CD47 mAb.
  • SIRPa and increase phagocytosis of human tumor cells.
  • “Phagocytosis” of cancer cells refers to the engulfment and digestion of such cells by macrophages, and the eventual digestion or degradation of these cancer cells and the release of digested or degraded cellular components extracellularly, or intracellularly to undergo further processing.
  • Anti-CD47 monoclonal antibodies that block SIRPa binding to CD47 increase macrophage phagocytosis of cancer cells. SIRPa binding to CD47 on cancer cells would otherwise allow these cells to escape macrophage phagocytosis.
  • the cancer cell may be viable or living cancer cells.
  • soluble anti-CD47 mAbs initiate a cell death program on binding to CD47 on tumor cells, resulting in collapse of mitochrondrial membrane potential, loss of ATP generating capacity, increased cell surface expression of phosphatidylserine (detected by increased staining for annexin V) and cell death without the participation of caspases or fragmentation of DNA.
  • Such soluble anti-CD47 mAbs have the potential to treat a variety of solid and hematological cancers.
  • soluble anti-CD47 mAbs which have been shown to induce tumor cell death, including MABL-1, MABL-2 and fragments thereof (US Patent 8, 101,719; Uno et al. Oncol Rep.
  • inducing cell death or “kills” and the like, are used interchangeably herein to mean that addition of an antibody compound of the present disclosure to cultured cancer cells causes these cells to display quantifiable characteristics associated with cell death including any one, or more, of the following:
  • Induction of cell death refers to the ability of certain of the soluble anti-CD47 antibodies, murine antibodies, chimeric antibodies, humanized antibodies, or antigen-binding fragments thereof (and competing antibodies and antigen-binding fragments thereof) disclosed herein to kill cancer cells via a cell autonomous mechanism without participation of complement or other cells including, but not limited to, T cells, neutrophils, natural killer cells, macrophages, or dendritic cells.
  • induction of cell death includes, but is not limited to, a greater than 2-fold increase in annexin V staining of human tumor cells caused by soluble anti-CD47 mAb compared to the background obtained with the negative control antibody (humanized, isotype-matched antibody).
  • Cell viability assays are described in NCI/NIH guidance manual that describes numerous types of cell based assays that can be used to assess induction of cell death caused by CD47 antibodies: "Cell Viability Assays", Terry L Riss, PhD, Richard A Moravec, BS, Andrew L Niles, MS, Helene A Benink, PhD, Tracy J Worzella, MS, and Lisa Minor, PhD. Contributor Information, published May 1, 2013.
  • CD47 is expressed on human erythrocytes (hRBCs) (Brown. J Cell Biol. I l l : 2785-2794, 1990; Avent. Biochem J, (1988) 251 : 499-505; Knapp. Blood, (1989) Vol. 74, No. 4, 1448-1450; Oliveira et al. Biochimica et Biophysica Acta 1818: 481-490, 2012; Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271). It has been shown that anti-CD47 mAbs bind to RBCs, including B6H12 (Brown et al. J.
  • Binding to RBCs can be reduced by generation of bi-specific antibodies with only one CD47 binding arm (Masternak et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 2482).
  • the terms “reduced binding to hRBCs”, refers to the Kd of an anti-CD47 mAb binding to a hRBC which is 10-fold or greater than the Kd on a human tumor cell, wherein the tumor cell is an O VI 0hCD47 cell.
  • no binding refers to no measurable binding to hRBCs at an anti-CD47 mAb concentration up to and including 100 ⁇ g/ml.
  • Some of the anti-CD47 mAbs, disclosed herein, have reduced or no detectable binding to human RBCs.
  • Red blood cell (RBC) agglutination or hemagglutination is a homotypic interaction that occurs when RBCs aggregate or clump together following incubation with various agents, including antibodies to RBC antigens and cell surface proteins such as CD47.
  • RBC antigens and cell surface proteins such as CD47.
  • Many anti-CD47 antibodies have been reported to cause hemagglutination of isolated human RBCs in vitro, in a concentration dependent manner, including B6H12, BRIC126, MABL-1, MABL-2, CC2C6, and 5F9 (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract nr 5011, US Patent 9,045,541, Uno et al. Oncol Rep.
  • the mouse antibody 2D3 is an example of an anti- CD47 antibody that binds to CD47 on red blood cells but does not cause hemagglutination (US Patent 9,045,541, Petrova et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271).
  • Hemagglutination has been shown to be reduced/eliminated by reducing the binding selectively to human RBCs, but not other cells, using a SIRPa-Fc fusion protein (Uger R. et al. Blood 2013; 122(21): 3935).
  • mouse anti-CD47 mAb 2A1 and humanized versions of 2A1 have been reported to block CD47/SIRPa but do not exhibit hemagglutination activity (US Patent 9,045,541).
  • a small number of a panel of mouse anti-human CD47 antibodies (3 of 23) were reported to not cause hemagglutination of human RBCs (Pietsch E et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 2470).
  • agglutination refers to cellular clumping
  • hemagglutination refers to clumping of a specific subset of cells, i.e., RBCs.
  • hemagglutination is a type of agglutination.
  • reduced hemagglutination refers to measurable agglutination activity of hRBCs at anti-CD47 mAb concentrations greater that 1.85 ⁇ g/ml, and no measurable activity at concentrations less than or equal to 1.85 ⁇ g/ml.
  • no detectable hemagglutination refers to no measurable agglutination activity of hRBCs at anti-CD47 mAb concentrations greater or equal to 0.3 pg/mL to a concentration less than or equal to 50 ⁇ g/mL.
  • Some of the anti-CD47 antibodies described herein cause reduced or no detectable hemagglutination of human RBCs.
  • TSP1 is also a ligand for CD47.
  • the TSP1/CD47 pathway opposes the beneficial effects of the NO pathway in many cell types, including, but not limited to, vascular cells.
  • the NO pathway consists of any of three enzymes (nitric oxide synthases, NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced, or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP).
  • cGMP messenger molecule cyclic GMP
  • the proper functioning of the NO/cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and IRI.
  • the inhibition of the NO/cGMP pathway by the TSP1/CD47 system exacerbates the effects of stress. This is a particular problem in the cardiovascular system where both cGMP and cAMP play important protective roles.
  • ischemia and reperfusion injury cause or contribute to disease, trauma, and poor outcomes of surgical procedures.
  • one of more of the chimeric or humanized anti-CD47 antibodies will reverse TSP1 inhibition of cGMP production. Reversal will be complete (>80 %) or intermediate (20% -80%).
  • immediate reversal of NO pathway inhibition refers to 20-80%> reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAb compared to a negative control, humanized isotype-matched antibody.
  • no reversal of NO pathway inhibition refers to less than 20% reversal of TSP1 inhibition of NO signaling by an anti-CD47 mAb compared to a negative control, humanized isotype-matched antibody.
  • Anti-CD47 mAbs exist in the prior art with combinations of some, but not all, of the functional characteristics described herein. Previously, it has been shown that humanized anti- CD47 mAbs such as AB6.12 IgGl, AB6.12-IgG4P, and AB6.12-IgG4PE (US Patent 9,045,541, US Patent Publication 2014/0161799, WO Publication 2014/093678, US Patent Publication 2014/0363442) and 5F9 (Mounho-Zamora B. et al. The Toxicologist, Supplement to Toxicological Sciences, 2015; 144 (1): Abstract 596: 127, Liu et al. PLoS One.
  • humanized anti- CD47 mAbs such as AB6.12 IgGl, AB6.12-IgG4P, and AB6.12-IgG4PE (US Patent 9,045,541, US Patent Publication 2014/0161799, WO Publication 2014/093678,
  • the humanized CD47 mAbs AB6.12 IgGl, AB6.12-IgG4P, and AB6.12-IgG4PE also do not cause hemagglutination of human RBCs (US Patent 9,045,541).
  • the 5F9 humanized anti-CD47 mAb binds to and causes hemagglutination of human RBCs (Uger R. et al. Cancer Res 2014; 74(19 Suppl): Abstract nr 5011, Sikic B. et al. J Clin Oncol 2016;34 (suppl; abstract 3019).
  • Murine anti-CD47 mAbs B6H12, BRIC126, and CC2C6 block the interaction of CD47 and SIRPa, cause phagocytosis, and bind to and cause hemagglutination of human RBCs (Petrova P. et al. Cancer Res 2015; 75(15 Suppl): Abstract nr 4271, Seiffert et al. Blood 94:3633-3643,1999; Vernon-Wilson et al. Eur J Immunol. 30: 2130-2137, 2000; Latour et al. J. Immunol. 167: 2547- 2554, 2001; Subramanian et al. Blood 107: 2548-2556, 2006; Liu et al.
  • Murine anti-CD47 mAbs MABL-1 and MABL-2 bind to human CD47, induce tumor cell death and cause RBC hemagglutination (US Patent 8,101,719); murine mAb Ad22 binds to human CD47 and induces tumor cell death (Pettersen et al. J. Immunol. 166: 4931-4942, 2001; Lamy et al. J Biol Chem. 278: 23915-23921, 2003); and murine mAb 1F7 binds to human CD47, blocks the interaction of CD47 and SIRPa and induces tumor cell death (Rebres et al. J. Cellular Physiol. 205: 182-193, 2005; Manna et al. J. Immunol. 170: 3544-3553, 2003; Manna et al. Cancer Research, 64: 1026-1036, 2004).
  • the preferred anti-CD47 antibodies described herein are also characterized by combinations of properties which are not exhibited by prior art anti-CD47 antibodies proposed for human therapeutic use. Accordingly, the preferred anti-CD47 antibodies described herein are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the anti-CD47 antibodies are characterized by:
  • hRBCs human red blood cells
  • the monoclonal antibody, or antigen binding fragment thereof also specifically binds to non-human primate CD47, wherein non-human primate may include, but is not limited to, cynomolgus monkey, green monkey, rhesus monkey and squirrel monkey.
  • the monoclonal antibody, or antigen binding fragment thereof binds human, non-human primate, mouse, rabbit, and rat CD47.
  • anti-CD47 mAbs with distinct functional profiles. These antibodies possess distinct combinations of properties selected from the following: 1) exhibit cross-reactivity with one or more species homologs of CD47; 2) block the interaction between CD47 and its ligand SIRPa; 3) increase phagocytosis of human tumor cells, 4) induce death of susceptible human tumor cells; 5) do not induce cell death of human tumor cells; 6) have reduced binding to human red blood cells (hRBCs); 7) have no detectable binding to hRBCs; 8) cause reduced agglutination of hRBCs; 9) cause no detectable agglutination of hRBCs; 10) reverse TSP1 inhibition of the nitric oxide (NO) pathway and/or 11) do not reverse TSP1 inhibition of the NO pathway.
  • NO nitric oxide
  • CD47 cell surface expression of CD47 and those expressing the highest levels of CD47 are appear to be the most aggressive and the most lethal for patients.
  • Increased CD47 expression is thought to protect cancer cells from phagocytic clearance by sending a "don't eat me” signal to macrophages via SIRPa, an inhibitory receptor that prevents phagocytosis of CD47-bearing cells (Oldenborg et al. Science 288: 2051-2054, 2000; Jaiswal et al. (2009) Cell 138(2):271-851; Chao et al. (2010) Science Translational Medicine 2(63):63ra94).
  • SIRPa an inhibitory receptor that prevents phagocytosis of CD47-bearing cells
  • Antibodies that block CD47 and prevent its binding to SIRPa have shown efficacy in human tumor in murine (xenograft) tumor models.
  • Such blocking anti-CD47 mAbs exhibiting this property increase the phagocytosis of cancer cells by macrophages, which can reduce tumor burden (Majeti et al. (2009) Cell 138 (2): 286-99; US 9,045,541; Willingham et al. (2012) Proc Natl Acad. Sci. USA 109(17):6662-6667; Xiao et al. (2015) Cancer Letters 360:302-309; Chao et al. (2012) Cell 142:699-713; Kim et al.
  • Anti-CD47 mAb Ad22 induces cell death of multiple human tumor cells lines (Pettersen et al. J. Immuno. 166: 4931-4942, 2001; Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003). AD22 was shown to indice rapid mitochondrial dysfunction and rapid cell death with early phosphatidylserine exposure and a drop in mitochondrial membrane potential (Lamy et al. J. Biol. Chem. 278: 23915-23921, 2003).
  • Anti- CD47 mAb MABL-2 and fragments thereof induce cell death of human leukemia cell lines, but not normal cells in vitro and had an anti-tumor effect in in vivo xenograft models. (Uno et al. (2007) Oncol. Rep. 17 (5): 1189-94).
  • Anti-human CD47 mAb 1F7 induces cell death of human T cell leukemias (Manna and Frazier (2003) J. Immunol. 170: 3544-53) and several breast cancers (Manna and Frazier (2004) Cancer Research 64 (3): 1026-36). 1F7 kills CD47-bearing tumor cells without the action of complement or cell mediated killing by NK cells, T cells, or macrophages.
  • anti-CD47 mAb 1F7 acts via a non-apoptotic mechanism that involves a direct CD47- dependent attack on mitochondria, discharging their membrane potential and destroying the ATP- generating capacity of the cell leading to rapid cell death. It is noteworthy that anti-CD47 mAb 1F7 does not kill resting leukocytes, which also express CD47, but only those cells that are "activated” by transformation. Thus, normal circulating cells, many of which express CD47, are spared while cancer cells are selectively killed by the tumor-toxic CD47 mAb (Manna and Frazier (2003) J. Immunol. 170: 3544-53).
  • mAb 1F7 also blocks binding of SIRPa to CD47 (Rebres et al et al. J. Cellular Physiol. 205: 182-193, 2005) and thus it can act via two mechanisms: (1) direct tumor toxicity, and (2) causing phagocytosis of cancer cells.
  • a single mAb that can accomplish both functions may be superior to one that only blocks CD47/SIRPa binding.
  • IRI ischemia-reperfusion injury
  • IRI contributes to poor outcomes in many surgical procedures where IRI occurs due to the necessity to stop blood flow for a period of time, in many forms/causes of trauma in which blood flow is interrupted and later restored by therapeutic intervention and in procedures required for organ transplantation, cardio/pulmonary bypass procedures, reattachment of severed body parts, reconstructive and cosmetic surgeries and other situations involving stopping and restarting blood flow.
  • Ischemia itself causes many physiological changes that, by themselves would eventually lead to cell and tissue necrosis and death.
  • Reperfusion poses its own set of damaging events including generation of reactive oxygen species, thrombosis, inflammation and cytokine mediated damage.
  • TSP1-CD47 The pathways that are limited by the TSP1-CD47 system are precisely those that would be of most benefit in combating the damage of IRI, including the NO pathway. Thus, blocking the TSP1-CD47 pathway, as with the antibodies disclosed herein, will provide more robust functioning of these endogenous protective pathways.
  • Anti-CD47 mAbs have been shown to reduce organ damage in rodent models of renal warm ishchemia (Rogers et al. J Am Soc Nephrol. 23: 1538-1550, 2012), liver ischemia-reperfusion injury (Isenberg et al. Surgery. ⁇ 44: 752-761, 2008), renal transplantation (Lin et al. Transplantation. 98: 394-401, 2014; Rogers et al. Kidney Interantional.
  • liver transplantation including steatotic livers (Xiao et al. Liver Transpl. 21 : 468-477, 2015; Xiao et al. Transplantation. 100: 1480-1489, 2016).
  • anti-CD47 mAb caused significant reductions of right ventricular systolic pressure and right ventricular hypertrophy in the monocrotaline model of pulmonary arterial hypertension (Bauer et al. Cardiovasc Res. 93: 682-693, 2012).
  • Studies in skin flap models have shown that modulation of CD47, including with anti-CD47 mAbs, inhibits TSP1 -mediated CD47 signaling.
  • Anti-CD47 mAbs have also been shown to be efficacious in models of other cardiovascular diseases.
  • anti-CD47 mAb mitigated cardiac myocyte hypertrophy, decreased left ventricular fibrosis, prevented an increase in left ventricular weight, decreased ventricular stiffness, and normalized changes in the pressure volume loop profile (Sharifi-Sanjani et al. J Am Heart Assoc. , 2014).
  • An anti- CD47 mAb ameliorated atherosclerosis in multiple mouse models (Kojima et al. Nature., 2016). Cancer Indications
  • anti-CD47 mAbs and antigen binding fragments thereof effective as cancer therapeutics which can be administered to patients, preferably parenterally, with susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, including systemic mastocytosis, acute lymphocytic (lymphoblastic) leukemia (ALL), T cell - ALL, acute myeloid leukemia (AML), myelogenous leukemia, chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), myeloproliferative disorder / neoplasm, monocytic cell leukemia, and plasma cell leukemia; multiple myeloma (MM); Waldenstrom's Macroglobulinemia; lymphomas, including histiocytic lymphoma and T cell lymphoma, B cell lymphomas, including Hodgkin's lymphoma and non-Hodgkin's lymphoma, such as low grade/folli
  • combination therapies are often employed in cancer treatment as single-agent therapies or procedures may not be sufficient to treat or cure the disease or condition.
  • Conventional cancer treatments often involve surgery, radiation treatment, the administration of a combination of cytotoxic drugs to achieve additive or synergistic effects, and combinations of any or all of these approaches.
  • chemotherapeutic and biologic therapy combinations employ drugs that work via different mechanisms of action, increasing cancer cell control or killing, increasing the ability of the immune system to control cancer cell growth, reducing the likelihood of drug resistance during therapy, and minimizing possible overlapping toxicities by permitting the use of reduced doses of individual drugs.
  • Classes of conventional anti-tumor/anti-neoplastic agents useful in the combination therapies encompassed by the present methods are disclosed, for example, in Goodman & Oilman 's The Pharmacological Basis of Therapeutics, Twelfth Edition (2010) L.L. Brunton, B.A. Chabner, and B. C. Knollmann Eds., Section VIII, "Chemotherapy of Neoplastic Diseases", Chapters 60-63, pp. 1665-1770, McGraw-Hill, NY, and include, for example, alkylating agents, antimetabolites, natural products, a variety of miscellaneous agents, hormones and antagonists, targeted drugs, monoclonal antibodies and other protein therapeutics.
  • the methods of the present disclosure are related to treatment of cancer indications and further comprises treating the patient via surgery, radiation, and/or administering to a patient in need thereof an effective amount of a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions.
  • a chemical small molecule or biologic drug including, but not limited to, a peptide, polypeptide, protein, nucleic acid therapeutic, conventionally used or currently being developed, to treat tumorous conditions.
  • the therapeutic methods disclosed and claimed herein include the use of the antibodies disclosed herein alone, and/or in combinations with one another, and/or with antigen-binding fragments thereof of the present disclosure that bind to CD47, and/or with competing antibodies exhibiting appropriate biological/therapeutic activity, as well, for example, all possible combinations of these antibody compounds to achieve the greatest treatment efficacy.
  • the present therapeutic methods also encompass the use of these antibodies, antigen-binding fragments thereof, competing antibodies and combinations thereof further in combination with: (1) any one or more anti-tumor therapeutic treatments selected from surgery, radiation, anti-tumor, anti -neoplastic agents, and combinations of any of these, or (2) any one or more of anti-tumor biological agents, or (3) equivalents of any of the foregoing of (1) or (2) as would be apparent to one of ordinary skill in the art, in appropriate combination(s) to achieve the desired therapeutic treatment effect for the particular indication.
  • Antibody and small molecule drugs that increase the immune response to cancer by modulating co-stimulatory or inhibitory interactions that influence the T cell response to tumor antigens are also of particular interest in the context of the combination therapeutic methods encompassed herein and include, but are not limited to, other anti-CD47 antibodies.
  • Administration of therapeutic agents that bind to the CD47 protein for example, antibodies or small molecules that bind to CD47 and prevent interaction between CD47 and SIRPa, are administered to a patient, causing the clearance of cancer cells via phagocytosis.
  • the therapeutic agent that binds to the CD47 protein is combined with a therapeutic agent such as an antibody, a chemical small molecule or biologic drug disclosed herein, directed against one or more additional cellular targets of CD70 (Cluster of Differentiation 70), CD200 (OX-2 membrane glycoprotein, Cluster of Differentiation 200), CD 154 (Cluster of Differentiation 154, CD40L, CD40 ligand, Cluster of Differentiation 40 ligand), CD223 (Lymphocyte-activation gene 3, LAG3, Cluster of Differentiation 223), KIR (Killer-cell immunoglobulin-like receptors), GITR (TNFRSF18, glucocorticoid-induced TNFR- related protein, activation-inducible T FR family receptor, AITR, Tumor necrosis factor receptor superfamily member 18), CD28 (Cluster of Differentiation 28), CD40 (Cluster of Differentiation 40, Bp50, CDW40, T FRSF5, Tumor necrosis factor receptor superfamily member 5, p50), CD86 (
  • Additional agents include IL-10 (Interleukin-10, human cytokine synthesis inhibitory factor, CSIF) and Galectins.
  • IL-10 Interleukin-10, human cytokine synthesis inhibitory factor, CSIF
  • YERVOY ipilimumab; Bristol-Meyers Squibb
  • YERVOY ipilimumab; Bristol-Meyers Squibb
  • KEYTRUDA ® pembrolizumab; Merck
  • OPDIVO ® nivolumab; Bristol-Meyers Squibb Company
  • TECENTRIQTM (atezolizumab; Roche) is an example of an approved anti-PD-Ll antibody.
  • IRI Ischemia-Reperfusion Injury
  • a CD47 mAb or antigen binding fragment thereof disclosed herein can be used to treat a number of diseases and conditions in which IRI is a contributing feature, and to treat various autoimmune, autoinflammatory, inflammatory and cardiovascular diseases.
  • diseases and conditions include: organ transplantation in which a mAb or antigen binding fragment thereof of the present invention is administered to the donor prior to organ harvest, to the harvested donor organ in the organ preservation solution, to the recipient patient, or to any combination thereof; skin grafting; surgical resections or tissue reconstruction in which such mAb or fragment is administered either locally by injection to the affected tissue or parenterally to the patient; reattachment of body parts; treatment of traumatic injury; pulmonary hypertension; pulmonary arterial hypertension; sickle cell disease (crisis); myocardial infarction; cerebrovascular disease; stroke; surgically-induced ischemia; acute kidney disease/kidney failure; any other condition in which IRI occurs and contributes to the pathogenesis of disease; autoimmune and inflammatory diseases, including arthritis, rheumatoi
  • Anti-CD47 mAbs and antigen binding fragments thereof of the present invention can also be used to increase tissue perfusion in a subject in need of such treatment.
  • Such subjects can be identified by diagnostic procedures indicating a need for increased tissue perfusion.
  • the need for increased tissue perfusion may arise because the subject has had, is having, or will have, a surgery selected from integument surgery, soft tissue surgery, composite tissue surgery, skin graft surgery, resection of a solid organ, organ transplant surgery, or reattachment or an appendage or other body part.
  • IRI Ischemia-Reperfusion Injury
  • the methods of the present disclosure can further comprise administering to a patient in need thereof an effective amount of therapeutic agent that binds to the CD47 protein and a nitric oxide donor, precursor, or both; a nitric oxide generating topical agent; an agent that activates soluble guanylyl cyclase; an agent that inhibits cyclic nucleotide phosphodiesterases; or any combination of any of the foregoing.
  • the nitric oxide donor or precursor can be selected from NO gas, isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil ® , and arginine.
  • NO gas isosorbide dinitrate, nitrite, nitroprusside, nitroglycerin, 3-Morpholinosydnonimine (SIN-1), S- nitroso-N-acetylpenicillamine (SNAP), Diethylenetriamine/NO (DETA/NO), S-nitrosothiols, Bidil ® , and arginine.
  • the agent that activates soluble guanylyl cyclase can be a non-NO (nitric oxide)-based chemical activator of soluble guanylyl cyclase that increases cGMP levels in vascular cells.
  • Such agents bind soluble guanylyl cyclase in a region other than the NO binding motif, and activate the enzyme regardless of local NO or reactive oxygen stress (ROS).
  • ROS reactive oxygen stress
  • Non-limiting examples of chemical activators of soluble guanylyl cyclase include organic nitrates (Artz et al. (2002) J. Biol. Chem. 277: 18253-18256); protoporphyrin IX (Ignarro et al. (1982) Proc. Natl. Acad. Sci.
  • the agent that inhibits cyclic nucleotide phosphodiesterases can be selected from, tadalafil, vardenafil, udenafil, sildenafil and avanafil. Treatment of Autoimmune, Autoinflammatory, Inflammatory Diseases and Cardiovascular Diseases
  • a therapeutic agent that binds to the CD47 protein for the treatment of an autoimmune, autoinflammatory, inflammatory disease and / or cardiovascular disease can be combined with one or more therapeutic agent(s) such as an antibody, a chemical small molecule, or biologic or a medical or surgical procedure which include, but are not limited to the following.
  • the combined therapeutic agents are: hydroxychloroquine, leflunomide, methotrexate, minocycline, sulfasalazine, abatacept, rituximab, tocilizumab, anti-TNF inhibitors or blockers (adalimumab, etanercept, infliximab, certolizumab pegol, golimumab), non-steroidal anti-inflammatory drugs, glucocorticoids, corticosteroids, intravenous immunoglobulin, anakinra, canakinumab, rilonacept, cyclophosphamide, mycophenolate mofetil, azathioprine, 6-mercaptopurine, belimumab, beta interferons, glatiramer acetate, dimethyl fumarate, fingolimod, teriflunomide, natalizumab, 5- aminosalicylic acid,
  • the combined therapeutic agents or procedures are: medical procedures and/or surgery, including percutaneous coronary intervention (coronary angioplasty and stenting), coronary artery bypass grafting, and carotid endarterectomy; therapeutic agents, including angiotensin-converting enzyme (ACE) inhibitors (including ramipril, quinapril, captopril, and enalapril), calcium channel blockers (including amiodipine, nifedipine, verapamil, felodipine and diltiazem), angiotensin-receptor blockers (including eposartan, olmesarten, azilsartan, valsartan, telmisartan, losartan, candesartan, and irbesartan), the combination of ezetimibe and simvastatin, PCSK9 inhibitors (including alirocumab and evolocumab), anacetrapib, and FDVIG-Co
  • ACE angiotens
  • the combined therapeutic agents are: ACE inhibitors, angiotensin receptor blockers, angiotensin receptor neprilsyn inhibitors (including the combination of sacubitril and valsartan), diuretics, digoxin, inotropes, beta blockers and aldosterone antagonists.
  • the combined therapeutic agents are: sildenafil, tadalafil, ambrisentan, bosentan, macitentan, riociguat, treprostinil, epoprostenol, iloprost, and selexipag.
  • the anti-CD47 mAb is administered before, at the same time or after the combined therapeutic agents or medical or surgical procedures.
  • Another useful class of compounds for the combination therapies contemplated herein includes modulators of SIRPa/CD47 binding such as antibodies to SIRPa, as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRPa to CD47.
  • modulators of SIRPa/CD47 binding such as antibodies to SIRPa, as well as soluble protein fragments of this ligand, or CD47 itself, inhibiting binding of, or interfering with binding of, SIRPa to CD47.
  • the therapeutic methods encompassed herein include the use of the antibodies disclosed herein alone, in combination with one another, and/or with antigen-binding fragments thereof as well, for example, all possible combinations of these antibody compounds.
  • Diagnostics have been an area of focus in the field of oncology.
  • a number of diagnostic assays have been developed for targeted therapeutics such as Herceptin (Genentech), Tarceva (OSI Pharmaceuticals / Genentech), Iressa (Astra Zeneca), and Erbitux (Imclone / Bristol Myers Squibb).
  • the anti-CD47 mAbs antibodies of the disclosure are particularly well-suited to use in diagnostic applications. Accordingly, the disclosure provides a method to measure CD47 expression in tumor and / or immune cells, using an anti-CD47 mAb of the disclosure.
  • the anti-CD47 mAbs of the disclosure may be used in a diagnostic assay and / or in vitro method to measure CD47 expression in tumor and / or immune cells present in a patient's tumor sample.
  • the anti-CD47 mAbs of the disclosure may bind CD47 on approximately 1% or more of tumor and / or immune cells present in a patient' s sample as compared to a reference level.
  • the anti-CD47 mAbs may bind CD47 on approximately 5% or more of tumor and / or immune cells in a patient's sample as compared to a reference level, for example, or binding at least 10%, or at least 20%, or at least 30%, or at least about 40%, or at least about 50%), or at least about 60%>, or at least about 70%, or at least about 80%, or at least about 90%, or between 10-100%) as compared to a reference level.
  • the anti-CD47 mAbs may bind CD47 on tumor and / or immune cells in a patient's sample to at least about a 2- fold increase as compared to a reference level, or at least about 3 -fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or between 2-fold and 10-fold or greater as compared to a reference level.
  • the measurement of CD47 expression in a patient's sample provides biological and/or clinical information that enables decision making about the development and use of a potential drug therapy, notably the use of anti-CD47 antibodies for treating solid and hematological cancers, autoimmune disease, inflammatory disease, atherosclerosis, heart failure, in which the CD47 receptor plays a role.
  • the in vitro method comprises, obtaining a patient sample, contacting the patient sample with a monoclonal antibody, or antigen-binding fragment thereof, which specifically binds to an epitope within the sequence of SEQ ID NO:65, and assaying for binding of the antibody to the patient sample, wherein binding of the antibody to the patient sample is diagnostic of CD47 expression in a patient sample.
  • the preferred CD47 antibodies, or antigen binding fragments thereof, for the in vitro method are those comprising a combination of a heavy chain (HC) and a light chain (LC), listed from the combination of:
  • VH amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto and the a VL amino acid sequence is at least 90%, 95%, 97%, 98% or 99% identical thereto.
  • a diagnostic assay in accordance with the disclosure may comprise contacting tumor and / or immune cells in a patient's sample with an anti-CD47 mAb, or an antigen binding fragment thereof, and assaying for binding of the anti-CD47 mAb to a patient's tumor sample, wherein binding of the anti-CD47 mAb to the patient sample is diagnostic of CD47 expression.
  • the patient's sample is a sample containing tumor cells.
  • binding of the anti-CD47 mAb of the disclosure, or antigen binding fragment thereof, to the tumor cells may be assessed for CD47 expression.
  • the levels of CD47 expression by tumor cells and/or immune cells of a patient' s tumor sample may be predictive of clinical outcome in a patient.
  • Increased binding of anti-CD47 mAbs binding to cells in a patient's sample is associated with increased CD47 expression.
  • the anti-CD47 mAbs of the disclosure may bind to approximately 5% or more of tumor cells in a patient's sample and this may indicate that the patient would benefit from rapid intervention to a solid and hematological cancer.
  • a diagnostic assay of this sort may be used to determine suitable therapeutic regimes for solid and hematological cancers with relatively high binding of anti-CD47 mAbs of the disclosure, i.e., increased CD47 expression.
  • the diagnostic assay of the disclosure may allow the user a greater deal of confidence in the CD47 expression in tumor and / or immune cells.
  • the increased sensitivity of the diagnostic assay of the disclosure allows detection of CD47 in a patient's sample at lower levels than has previously been the case.
  • the anti-CD47 mAbs of the disclosure may be used as a diagnostic assay in relation to many forms of cancer.
  • Particular forms of cancer that may advantageously be investigated for CD47 expression include susceptible hematologic cancers and solid tumors including, but not limited to, leukemias, lymphomas, and solid tumors.
  • the diagnostic assays of the disclosure may utilize any suitable means for detecting binding of an anti-CD47 mAb to measure CD47 expression. Suitable methods may be selected with reference to the nature of any reporter moiety used to label the anti-CD47 mAbs of the disclosure. Suitable techniques include, but are by no means limited to, flow cytometry, and enzyme linked immunosorbent assays (ELISA) and assays utilizing nanoparticles. It is particularly preferred that a diagnostic assay of the invention be one involving immunohistochemistry in which a tumor sample is exposed to an anti-CD47 mAb of the disclosure, and the level of cell labelling is assessed by immunohistochemistry.
  • VSS (SEQ ID NO:36).
  • Chimeric antibodies disclosed herein comprise a mouse heavy chain variable domain and a light chain variable domain combined with a human kappa or human Fc IgGl, IgGl-N297Q, IgG2, IgG4, IgG4 S228P, and IgG4 PE constant domains, respectively. These were designed to incorporate a secretion signal and cloned into a mammalian expression system, and transfected into CHO cells to generate chimeric (murine-human) antibodies. The chimeric variants were expressed as full length IgG molecules, secreted into the medium, and purified using protein A.
  • the humanized antibodies disclosed herein comprise frameworks derived from the human genome.
  • the collection covers the diversity found in the human germ line sequences, yielding functionally expressed antibodies in vivo.
  • the complementarity determining regions (CDRs) in the light and heavy chain variable regions of the murine and chimeric (murine-human) are described herein and were determined by following commonly accepted rules disclosed in "Protein Sequence and Structure Analysis of Antibody Variable Domains", In: Antibody Engineering Lab Manual, eds. S. Duebel and R. ontermann, Springer- Verlag, Heidelberg (2001)).
  • the human light chain variable domains were then designed.
  • the humanized variable domains were then combined with a secretion signal and human kappa and human Fc IgGl, IgGl- N297Q, IgG2, IgG3, IgG4 S228P and IgG4 PE constant domains, cloned into a mammalian expression system, and transfected into CHO cells to generate humanized mAbs.
  • the humanized variants were expressed as full length IgG molecules, secreted into the medium and purified using protein A.
  • IgGl-N297Q A non-glycosylated version (IgGl-N297Q) was created by site directed mutagenesis of heavy chain position 297 to change the asparagine to glutamine (Human Fc IgGl-N297Q, SEQ ID NO:54).
  • An IgG4 variant was created by site-directed mutagenesis at position 228 to change the serine to proline thereby preventing in vivo Fab arm exchange.
  • An IgG4 double mutant was created by site-directed mutagenesis at positions 228 (serine to proline) and 235 (leucine to glutamate) to prevent Fab arm exchange and to further reduce Fc effector function.
  • IgG2, IgG3, IgG4 S228P, and IgG4 PE isotypes were constructed by cloning the heavy chain variable domain in frame with the human IgG2, IgG3, IgG4 S228P, and IgG4PE constant domains (Human Fc- IgG2, SEQ ID NO:55 Human Fc-IgG3, SEQ ID NO:56; Human Fc-IgG4 S228P, SEQ ID NO:58; and Human Fc-IgG4 PE, SEQ ID NO:59).
  • chimeric (murine-human) and humanized antibodies of the present disclosure was determined by ELISA using OVIO cells transfected with human CD47 (OVIO hCD47) or using freshly isolated human red blood cells (hRBCs), which display CD47 on their surface (Kamel et al. (2010) Blood. Transfus. 8(4):260-266).
  • Binding activities of VLX4, VLX8, and VLX9 chimeric (murine-human) and humanized mAbs were determined using a cell-based ELISA assay with human OV10 hCD47cells expressing cell surface human CD47.
  • OV10 hCD47 cells were grown in IMDM medium containing 10% heat inactivated fetal bovine serum (BioWest; S01520).
  • 3xl0 4 cells were plated in 96 well cell bind plates (Corning #3300, VWR #66025-626) and were 95-100% confluent at the time of assay.
  • Binding activities of chimeric (murine-human) and humanized VLX4, VLX8, and VLX9 mAbs to human CD47 on hRBCs were also determined using flow cytometry.
  • hRBCs were incubated for 60 min on at 37°C with various concentrations of the chimeric or humanized antibodies in a solution of phosphate buffered saline, pH 7.2, 2.5 mM EDTA (PBS+E). Cells were then washed with cold PBS+E, and incubated for an additional hour on ice with FITC labeled donkey anti-human antibody (Jackson Immuno Research Labs, West Grove, PA; Catalogue # 709- 096-149) in PBS +E.
  • VLX8 humanized mAbs VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE, VLX8hum_04 IgG4 PE, VLX8hum_05 IgG4 PE, and VLX8hum_06 IgG4 PE, VLX8hum_07 IgG4 PE, VLX8hum_08 IgG4 PE, VLX8hum_09 IgG4 PE, VLX8hum_l l IgG4 PE, VLX8hum_06 IgG2, VLX8hum_07 IgG2, VLX8hum_08 and VLX8hum_09 IgG2 IgG2 bound to human RBCs with Kd values similar to the values obtained for OV10 hCD47 tumor cells whereas VLX8hum_10 IgG4 PE exhibited reduced, but measurable binding to hRBCs (FIG.
  • Table 1 shows the apparent binding affinities of VLX9 murine-human chimeric mAbs to human OV10 hCD47 cells and to human RBCs. All of the chimeric mAbs bound to OV10 hCD47 tumor cells with apparent Kd values in the picomolar range. Similarly, the humanized VLX9 mAbs bound to human OV10 hCD47 tumor cells in a concentration-dependent manner (FIG. 5 A and FIG. 5B) with apparent affinities in the picomolar to nanomolar range (Table 2).
  • VLX9 chimeric mAbs bound to hRBCs with apparent Kd values in the picomolar range and these were similar to the Kd values obtained for OV10 hCD47 tumor cells by ELISA (Table 1).
  • VLX9 humanized mAbs VLX9hum_01 IgG2, VLX9hum_ 02 IgG2 and VLX9hum_07 IgG2 exhibited reduced but measurable binding to human RBCs (FIG. 6, Table 2).
  • Humanized mAbs VLX9hum_03, 04, 05, 06, 08, 09 and 10 IgG2 exhibited no measureable binding to RBCs (Table 2). This differential binding of the humanized mAbs to tumor cells and RBCs was unexpected as the VLX9 IgG2 chimeric mAbs all bound with similar apparent Kd values to both tumor and RBC CD47 (Table 1).
  • Table 1 Binding of VLX4. VLX8. and VLX9 Chimeric (xi) mAbs to OV10 hCD47 Cells and Human Red Blood Cells (hRBCs).
  • VLX4 VLX8. and VLX9 Humanized mAbs to Human OV10 hCD47 and Human Red Blood Cells (hRBCs).
  • Table 3 shows the apparent binding affinities of the humanized VLX4 and VLX8 mAbs to RBCs from mouse, rat, and cynomolgus monkey determined by non-linear fit (Prism GraphPad software) of the median fluorescence intensities at various antibody concentrations. This data demonstrates that humanized VLX4 and VLX8 mAbs bind to mouse, rat, rabbit (data not shown) and cynomolgus monkey RBCs with apparent Kd values in the picomolar to nanomolar range (Table 4).
  • CD47 Antibodies Block CD47/SIRPa binding
  • SIRPa-Fc fusion protein (R&D Systems, cat #4546-SA) was labelled using an Alexa Fluor® antibody labelling kit (Invitrogen Cat No. A20186) according to the manufacturers specifications.
  • 1.5 x 10 6 Jurkat T cells were incubated with humanized mAbs ⁇ g/ml), a human control antibody in RPMI containing 10% media or media alone for 30 min at 37°C.
  • An equal volume of fluorescently labeled SIRPa-Fc fusion protein was added and incubated for an additional 30 min at 37°C.
  • Cells were washed once with PBS and the amount of labelled SIRPa- Fc bound to the Jurkat T cells analyzed by flow cytometry.
  • the humanized VLX4, VLX8 and VLX9 mAbs blocked the interaction of CD47 expressed on the Jurkat T cells with SIPRa, while the human control antibody (which does not bind to CD47) or media alone, did not block the CD47/SIRPa interaction.
  • Human derived macrophages were derived from leukapheresis of healthy human peripheral blood and incubated in AIM-V media (Life Technologies) for 7-10 days.
  • AIM-V media Life Technologies
  • macrophages were re-plated at a concentration of lxlO 4 cells per well in 100 ul of AEVI-V media in a 96-well plate and allowed to adhere for 24 hrs.
  • the target human cancer cells (Jurkat) were labeled with ⁇ 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE; Sigma Aldrich) and added to the macrophage cultures at a concentration of 5xl0 4 cells in 1ml of AIM-V media (5: 1 target to effector ratio).
  • VLX4, VLX8, and VLX9 CD47 mAbs (1 ⁇ g/ml) were added immediately upon mixture of target and effector cells and allowed to incubate at 37°C for 2-3 hours.
  • VLX4 chimeric (murine-human) mAbs VLX4 IgGl, VLX4 IgGl N297Q, VLX4 IgG4 PE, and VLX4 IgG4 S228P increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRPa interaction and this enhanced phagocytosis is independent of Fc function.
  • VLX4hum_01 IgGl VLX4hum_01 IgG4
  • VLX4hum_06 IgG4 PE VLX4hum_07 IgG4 PE
  • VLX4hum_12 IgG4 PE VLX4hum_13 IgG4 PE
  • VLX4hum_13 IgG4 PE increased phagocytosis of Jurkat cells by human macrophages by blocking the CD47/SIRPa interaction.
  • VLX8 chimeric (murine-human) mAbs VLX8 IgGl N297Q and VLX8 IgG4 PE increase phagocytosis of Jurkat T cells by human macrophages via blocking the CD47/SIRPa interaction and this enhanced phagocytosis is independent of Fc function.
  • VLX8hum_01 IgG4 PE, VLX8hum_03 IgG4 PE, VLX8hum_07 IgG4 PE, VLX8hum_08 IgG4 PE, and VLX8hum_09 IgG4 PE increased phagocytosis of Jurkat cells by human macrophage by blocking the CD47/SIRPa interaction and this enhanced phagocytosis is independent of Fc function
  • VLX9 IgGlN297Q, VLX9 IgG2 and VLX9 IgG4 PE chimeric mAbs all increased phagocytosis of Jurkat T cells by human macrophages by blocking the CD47/SIRPa interaction and this enhanced phagocytosis is independent of Fc effector function.
  • FIG. 1 IB all of the humanized VLX9 IgG2 mAbs increased phagocytosis of Jurkat T cells.
  • Some soluble CD47 antibodies have been shown to induce selective cell death of tumor cells. This additional property of selective toxicity to cancer cells is expected to have advantages compared to mAbs that only block SIRPa binding to CD47.
  • lxlO 5 transformed human T cells Jurkat T cells
  • VLX4, VLX8, and VLX9 CD47 mAbs ⁇ g/ml
  • telomeres were then stained with fluorescently labeled annexin V and PI or 7- aminoactinomycin D (7-AAD) (BD Biosciences) and the signal detected using an Accuri C6 flow cytometer (BD Biosciences).
  • the increase in PS exposure is determined by measuring the percent increase in annexin V signal and the percent of dead cells by measuring the percent increase in PI or 7-AAD signal.
  • these mAbs induce cell death of tumor cells directly and do not require complement or the intervention of other cells (e.g., NK cells, T cells, or macrophages) to kill.
  • the mechanism is independent of both other cells and of Fc effector function. Therefore, therapeutic antibodies developed from these mAbs can be engineered to reduce Fc effector functions such as ADCC and CDC and thereby limit the potential for side effects common to humanized mAbs with intact Fc effector functions.
  • the soluble VLX4 humanized mAbs induced cell death of Jurkat T ALL cells as measured by increased annexin V staining and 7-AAD staining (not shown).
  • the humanized mAbs VLX4hum_01 IgGl, VLX4hum_01 IgG4 PE, VLX4hum_02 IgGl, VLX4hum_02 IgG4 PE, VLX4hum_06 IgG4 PE, VLX4hum_07 IgG4 PE, VLX4hum_12 IgG4 PE, and VLX4hum_13 IgG4 PE caused cell death.
  • the humanized mAbs VLX4hum_08 IgG4 PE and VLX4hum_l 1 IgG4 PE did not cause cell death of Jurkat T cells. Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
  • the soluble VLX8 chimeric and humanized mAbs induced cell death of Jurkat T ALL cells as measured as measured by increased annexin V staining and 7-AAD staining (not shown).
  • the humanized mAbs VLX8hum_02 IgG4 PE and VLX8hum_04 IgG4 PE did not cause cell death of Jurkat T cells. Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
  • the soluble VLX9 chimeric antibodies induced cell death of Jurkat cells as measured by increased annexin V staining and 7-AAD staining (not shown).
  • the chimeric VLX9 IgG2xi mAb and the humanized mAbs VLX9hum_06 IgG2, VLX9hum_07 IgG2, VLX9hum_08 IgG2, and VLX9hum_09 IgG2 induced cell death of Jurkat cells (greater than 2-fold increase in annexin V staining).
  • VLX9hum_01 IgG2, VLX9hum_02 IgG2, VLX9hum_03 IgG2, VLX9hum_04 IgG2, VLX9hum_05 IgG2 and VLX9hum_010 IgG2 did not cause cell death of Jurkat cells. . Induction of cell death and the promotion of phagocytosis of susceptible cancer cells imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
  • Hemagglutination of hRBCs was assessed following incubation of hRBCs with various concentrations of chimeric and humanized VLX4, VLX8, and VLX9 mAbs in vitro essentially as described by Kikuchi et al. Biochem Biophys Res. Commun (2004) 315:912-918. Blood was obtained from healthy donors, diluted (1 :50) in PBS/1 mM EDTA/BSA and and washed 3 times with PB S/EDT A/B S A. hRBCs were added to U- bottomed 96 well plates with equal volumes of the antibodies (75 ⁇ of each) and incubated for 3 hrs at 37°C and overnight at 4°C.
  • VLX4hum_01 IgGl N297Q caused hemagglutination of hRBCs
  • humanized VLX4hum_01 IgG4 PE mAb did not (mAb concentrations 50 ⁇ g/ml to 0.3 ng/ml).
  • the lack of hemagglutination by VLX4hum_01 IgG4 PE imparts an additional desirable antibody property and therapeutic benefit in the treatment of cancer.
  • the chimeric antibody VLX8 IgG4 PE (xi) and the humanized antibodies VLX8hum_08 IgG4 PE, VLX8hum_09 IgG4 PE, and VLX8hum_010 IgG4 PE caused hemagglutination of hRBCs, whereas the VLX8 humanized Abs VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and VLX8hum_l 1 IgG4 PE did not (mAb concentrations 50 ⁇ g/ml to 0.3 ng/ml).
  • VLX4hum_01 IgG4 PE, VLX8hum_01 IgG4 PE, VLX8hum_02 IgG4 PE, VLX8hum_03 IgG4 PE and VLX8hum_l l IgG4 PE imparts an additional desirable antibody property and a therapeutic benefit in the treatment of cancer.
  • VLX9 IgG2 caused hemagglutination of hRBCs, whereas all of the humanized VLX9 mAbs except for VLX9hum_07 IgG2, did not (at concentrations from 50 ug/ml to 0.3 pg/ml). However, the amount of hemagglutination caused by VLX9hum_07 was reduced compared to the VLX9 IgG2 chimeric mAb. Again, the lack of hemagglutination by the VLX9 humanized mAbs imparts an additional desirable antibody property and a therapeutic benefit in the treatment of cancer.
  • VLX4, VLX8 and VLX9 humanized antibodies exemplified by VLX4 07 IgG4PE, VLX8 10 IgG4PE and VLX9hum_08 IgG2, reduce tumor burden in vivo in a mouse xenograft model of lymphoma.
  • Raji human Burkitt's lymphoma cells (ATCC #CCL-86, Manassas, VA) were maintained in RPMI-1640 (Lonza; Walkersville, MD) supplemented with 10% Fetal Bovine Serum (FBS; Omega Scientific; Tarzana, CA) within a 5% C02 atmosphere. Cultures were expanded in tissue culture flasks.
  • mice Female NSG (NOD-Cg-Prkdc scid I12rg tolwj VSzJ) were obtained from Jackson Laboratory (Bar Harbor, ME) at 5-6 weeks of age. Mice were acclimated prior to handling and housed in microisolator cages (Lab Products, Seaford, DE) under specific pathogen-free conditions. Mice were fed Teklad Global Diet ® 2920x irradiated laboratory animal diet (Envigo, Formerly Harlan; Indianapolis, IN) and provided autoclaved water ad libitum. All procedures were carried out under Institutional Animal Care and Use guidelines.
  • PE significantly reduced (p ⁇ 0.0001, two-way ANOVA) tumor growth of the Raji tumors, demonstrating anti-tumor efficacy in vivo.
  • VLX9hum_08 IgG2 or vehicle (PBS) was administered as a 1 hour intravenous infusion on day 1 at a dose of 5 mg/kg and on day 18 at a dose of 15 mg/kg (3 animals/group). Hematological parameters were measured throughout the study on days -7, -3, 3, 8, 12, 18 (pre-dose), 20, 25, 29, 35 and 41 and compared/normalized to the means values of control animals. The pre-treatment RBC and Hg values on day 0 in the VLX9hum_08 IgG2 group were lower than the control group.
  • CD47 expression was determined in formalin-fixed, paraffin embedded (FFPE) blocks from patients with a number of types of cancer (obtained from commercial sources) using mouse/rabbit chimeric anti-CD47 mAbs. 3-4 micron sections were cut from FFPE blocks, deparaffinized and treated with antigen retrieval solution. Sections were then incubated with 4 ⁇ g/ml of the primary anti-CD47 mouse/rabbit chimeric mAbs for 1 hr and with an anti-rabbit HRP labeled secondary antibody for 20 minutes. The anti-CD47 antibody bound to human CD47 was visualized using the peroxidase substrate, 3,3',5,5'-tetramethylbenzidene.
  • Sections were counterstained with hematoxylin and evaluated using standard light microscopy. As shown in FIG. 21, high CD47 expression was detected in human breast cancer tissue, as shown by dark areas denoted by arrows, using CD47 mouse/rabbit chimeric mAbs, exemplified by the VLX4 mouse/rabbit chimeric mAb. This demonstrates that these mAbs can be used for immunohistochemical localization of human CD47 in tumor tissue sections obtained from FFPE blocks in diagnostic assays.
  • TSP1 binding to CD47 activates the heterotrimeric G protein Gi, which leads to suppression of intracellular cyclic AMP (cAMP) levels.
  • TSP1/CD47 pathway opposes the beneficial effects of the nitric oxide (NO) pathway in all vascular cells.
  • NO nitric oxide
  • the NO pathway consists of any of three nitric oxide synthase enzymes (NOS I, NOS II and NOS III) that generate bioactive gas NO using arginine as a substrate. NO can act within the cell in which it is produced or in neighboring cells, to activate the enzyme soluble guanylyl cyclase that produces the messenger molecule cyclic GMP (cGMP).
  • the proper functioning of the NO/cGMP pathway is essential for protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI).
  • stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI).
  • IRI ischemia-reperfusion injury
  • humanized anti-CD47 mAbs of the present disclosure exhibit the ability to reverse TSPl-mediated inhibition of NO-stimulated cGMP synthesis as, for example, described previously using mouse monoclonal antibodies to CD47 as disclosed by Isenberg et al. (2006) J. Biol. Chem. 281 :26069-80, or alternatively other downstream markers of or effects resulting from NO signaling, for example smooth muscle cell relaxation or platelet aggregation as described previously by Miller et al. (2010) Br J. Pharmacol. 159: 1542-1547.
  • Cells will be grown in Iscove's modified Dulbeccco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # SO 1520), 100 units/mL penicillin, 100 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL.
  • Iscove's modified Dulbeccco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalogue # SO 1520), 100 units/mL penicillin, 100 ⁇ g mL streptomycin (Sigma; Catalogue # P4222) at densities less than 1 x 106 cells/mL.
  • cells will be plated in 96 well tissue culture plates at a density of lxlO 5 cells/ml in Iscoves modified Dulbecco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # SO 1520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222) for 24 hours and then transferred to serum free medium overnight.
  • Iscoves modified Dulbecco's medium containing 5% (v/v) heat inactivated fetal bovine serum (BioWest; Catalog # SO 1520), 100 units/mL penicillin, 100 ⁇ g/mL streptomycin (Sigma; #P4222) for 24 hours and then transferred to serum free medium overnight.
  • humanized antibodies as disclosed herein purified from transient transfections in CHO cells as described above in Example 3, as well as the control chimeric antibody, will then be added at a final concentration of 20 ng/ml, followed 15 minutes later by 0 or 1 ⁇ g/ml human TSP1 (Athens Research and Technology, Athens, GA, Catalogue # 16-20-201319). After an additional 15 minutes, the NO donor, diethylamine (DEA) NONOate (Cayman Chemical, Ann Arbor, MI, Catalog # 82100), will be added to half the wells at a final concentration of 1 ⁇ . Five minutes later, the cells will be lysed with buffer supplied in the cGMP kit, and aliquots of each well assayed for cGMP content.
  • DEA diethylamine
  • TSP1 inhibition of cGMP It is anticipated that some of the chimeric or humanized antibodies will reverse TSP1 inhibition of cGMP. Reversal will be complete (>80 %) or intermediate (20% -80%). This reversal of TSP1 inhibition of cGMP will demonstrate that they have the ability to increase NO signaling and suggest utility in protecting the cardiovascular system against stresses including, but not limited to, those resulting from wounding, inflammation, hypertension, metabolic syndrome, ischemia, and ischemia-reperfusion injury (IRI). Additional assay systems, for example smooth muscle cell contraction, will also be expected to show that some of the chimeric or humanized antibody clones reverse the inhibitory actions of TSP on downstream effects resulting from the activation of NO signaling.
  • IRI ischemia-reperfusion injury

Abstract

L'invention concerne des anticorps monoclonaux anti-CD47 (Acm anti-CD47) possédant des profils fonctionnels distincts tels que décrits ici, des procédés pour générer des Acm anti-CD47, et des procédés d'utilisation de ces Acm anti-CD47 en tant qu'agents thérapeutiques pour la prévention et le traitement de cancers solides et hématologiques, de lésion de reperfusion ischémique, de maladies cardiovasculaires, de maladies auto-immunes, de maladies inflammatoires ou en tant qu'agents de diagnostic pour déterminer le niveau de CD47 dans des échantillons de tissu.
PCT/US2016/052383 2015-09-18 2016-09-17 Anticorps cd47 thérapeutiques WO2017049251A2 (fr)

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JP2018514882A JP6885606B2 (ja) 2015-09-18 2016-09-17 治療用ｃｄ４７抗体
EP16847511.9A EP3349787A4 (fr) 2015-09-18 2016-09-17 Anticorps cd47 thérapeutiques
NZ740686A NZ740686A (en) 2015-09-18 2016-09-17 Therapeutic cd47 antibodies
AU2016324316A AU2016324316B2 (en) 2015-09-18 2016-09-17 Therapeutic CD47 antibodies
UAA201808714A UA126146C2 (uk) 2015-09-18 2016-09-17 Терапевтичне антитіло до cd47
CN202211086845.8A CN116425875A (zh) 2015-09-18 2016-09-17 治疗性cd47抗体
MX2018003374A MX2018003374A (es) 2015-09-18 2016-09-17 Anticuerpos terapeuticos cd47.
CA2998644A CA2998644A1 (fr) 2015-09-18 2016-09-17 Anticorps cd47 therapeutiques
RU2018124859A RU2748401C2 (ru) 2015-09-18 2016-09-17 Терапевтические антитела к CD47
BR112018005322-8A BR112018005322A2 (pt) 2015-09-18 2016-09-17 anticorpo monoclonal ou seu fragmento de ligação a antígenos, composição farmacêutica, anticorpo monoclonal ou seu fragmento de ligação a antígenos para uso, método de tratamento de lesão de isquemia-reperfusão, método de tratamento de câncer em um paciente humano, método de avaliação da expressão de cd47 em células tumorais e/ou imunes usando um anticorpo monoclonal ou seu fragmento de ligação a antígenos
CN201680062144.7A CN108348589B (zh) 2015-09-18 2016-09-17 治疗性cd47抗体
US15/820,054 US10239945B2 (en) 2015-09-18 2017-11-21 Therapeutic CD47 antibodies
PH12018500515A PH12018500515A1 (en) 2015-09-18 2018-03-09 Therapeutic cd47 antibodies
IL258168A IL258168A (en) 2015-09-18 2018-03-15 Therapeutic cd47 antibodies
ZA2018/04326A ZA201804326B (en) 2015-09-18 2018-06-27 Therapeutic cd47 antibodies
US16/271,513 US10844124B2 (en) 2015-09-18 2019-02-08 Therapeutic CD47 antibodies
US17/084,156 US20210070865A1 (en) 2015-09-18 2020-10-29 Therapeutic cd47 antibodies

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US201562221852P 2015-09-22 2015-09-22
US62/221,852 2015-09-22
US201562232681P 2015-09-25 2015-09-25
US62/232,681 2015-09-25
US201562252171P 2015-11-06 2015-11-06
US62/252,171 2015-11-06
US201562263544P 2015-12-04 2015-12-04
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